Oxalaticus OX1 Research Articles

Complete Genome Sequences of the Soil Oxalotrophic Bacterium Cupriavidus oxalaticus Strain Ox1 and Its Derived mCherry-Tagged Strain.

ABSTRACTHere, we report the complete genome sequences of the soil oxalotrophic bacterium Cupriavidus oxalaticus Ox1 and a derived mCherry-tagged strain. The genome size is approximately 6.69 Mb, with a GC content of 66.9%. The genome sequence of C. oxalaticus Ox1 contains a complete operon for the degradation and assimilation of oxalate.

In vivo cloning of a Pseudomonas oxalaticus σ54-like gene acting in expression of hydrogenase genes from Alcaligenes hydrogenophilus

Abstract In Pseudomonas oxalaticus OX1, which can express the hydrogenase genes from the H 2 -oxidizing bacterium Alcaligenes hydrogenophilus , a σ 54 -like factor-defective mutant was isolated by insertion of the kanamycin resistance marker. The P. oxalaticus OX1 chromosome gene complementing the mutation was cloned in vivo using the R-plasmid R68.45. The recombinant plasmids formed in strain OX1, the R68.45-prime carrying a σ 54 -like gene, were selected using intrastrain mating of strain OX1. Carrying one of the recombinant plasmids, the σ 54 -like factor-defective mutant recovered the abilities to express the hydrogenase genes and to assimilate nitrate as the nitrogen source and C 4 -dicarboxylates as the carbon sources for growth.

Identification of 4-hydroxyvaleric acid as a constituent of biosynthetic polyhydroxyalkanoic acids from bacteria

Twenty-four different strains of aerobic Gram-negative bacteria, mainly belonging to the genera Alcaligenes, Paracoccus, Pseudomonas and Methylobacterium, were examined with respect to their ability to utilize 4-hydroxyvaleric acid (4HV), 4-valerolactone (4VL) and 3-hydroxypropionic acid (3HP) as carbon sources for growth and for accumulation of polyhydroxyalkanoic acid (PHA). A gas chromatographic (GC) method for the detection of 3-hydroxyalkanoic acid methyl esters has been extended for the detection of derivatives obtained from the methanolysis of 4-hydroxybutyric acid (4HB) and 4HV. Most of the Alcaligenes species and P. oxalaticus Ox1 accumulated a terpolyester consisting of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV) and 4HV as constituents from 4HV or 4VL as sole carbon sources in batch, fed-batch or two-stage fed-batch cultures. Poly(3HB-co-3HV-co-4HV) accumulated from 4HV by A. eutrophus strain NCIB 11599 amounted to approximately 50% of the cell dry matter and was composed of 42.0 mol % 3HB, 52.2 mol % 3HV and 5.6 mol % 4HV, respectively. Pseudomonads, which belong to the rRNA homology group I, were not able to incorporate 4HV. With 3HP as carbon source, the GC analysis provided evidence for the presence of 3HP in the PHA of many bacteria. Nuclear magnetic resonance spectroscopic analysis confirmed that, for example, A. eutrophus strain TF93 accumulated poly(3HB-co-3HP) with 98 mol % 3HB and 2 mol % 3HP if the cells were cultivated in the presence of 0.5% (w/v) 3HP.

In vivo cloning of genes determining lithoautotrophy (Aut) on a plasmid from Alcaligenes hydrogenophilus

Hydrogenase ( hox) genes on the megaplasmid pHG21-a from Alcaligenes hydrogenophilus, whose lithoautotrophic growth (Aut) is supported by H 2-oxidation (Hox) and CO 2-fixation (Cfx), were cloned in vivo using a broad host range IncP1 plasmid R68.45. The recombinant plasmid was detected by the characteristic that it was transferred at a frequency 10 6-fold higher than pHG21-a in intrastrain mating of the Hox − Cfx + bacterium Pseudomonas oxalaticus OX1. All of six recombinant plasmids designated pFUs inherited all three resistance markers of R68.45. Four plasmids (pFU3, pFU8, pFU11, and pFU15) with a molecular size of 69 Md had only membrane-bound hydrogenase ( hoxP) genes, and two plasmids (pFU7 and pFU9) of 85 Md had both hoxP and soluble hydrogenase ( hoxS) genes. The Hox − Cfx − bacteria P. oxalaticus OX4 and OX6 gained Aut − phenotype by the possession of pHG21-a, pFU7 or pFU15. These results showed that Hox plasmid pHG21-a was an Aut plasmid and pFU7 and pFU15 inherited this phenotype, pFU7 was maintained stably in P. oxalaticus OX1 and had all of the lithoautotrophic phenotypes of pHG21-a. pFU7, rather than pHG21-a, is useful for further studies on the transfer of the Aut phenotype to a broad range of bacteria.

Thermodynamic efficiency of bacterial growth calculated from growth yield of Pseudomonas oxalaticus OX1 in the chemostat

In order to determine the thermodynamic efficiency of bacterial growth, Pseudomonas oxalaticus OX1 was grown in carbon-limited continuous cultures. 11 different carbon sources, ranging from oxalate (most oxidised component) to ethanol (most reduced component), were used as limiting substrate in these experiments. From the experimental yield values (expressed as C-mol dry weight produced per C-mol carbon substrate consumed) the thermodynamic efficiencies were calculated. On substrates more reduced than biomass (such as ethanol and glycerol) the thermodynamic efficiency of growth of P. oxalaticus was negative but it reached a maximum of 23 +/- 3% with substrates with a degree of reduction of 3 (citrate) and lower. The actual concentrations of the components involved were incorporated into the calculations but this affected the overall thermodynamic efficiency only to a small extent. This result strengthens the conclusion of Westerhoff et al. (Westerhoff, H.V., Hellingwerf, K.J. and Van Dam, K. (1983) Proc. Natl. Acad. Sci. 80, 305-309) that bacteria have been optimised towards a theoretical thermodynamic efficiency of 24%, corresponding with maximisation of growth rate at optimal efficiency, with highly oxidised substrates.

Transmissibility of hydrogen oxidation (Hox) plasmid from Alcaligenes hydrogenophilus

The transmissibility of the Hox plasmid pHG21-a, transferred from Alcaligenes hydrogenophilus to Pseudomonas oxalaticus OX1 and OX4 by conjugation, was examined in each recipient. Hox plasmid pHG21-a behaved as a self-transmissible plasmid in P. oxalaticus OX4 as in A. hydrogenophilus, but in P. oxalaticus OX1, it behaved as nonself-transmissible one, being mobilized by R-plasmids. It has been argued whether Hox plasmids of hydrogen-oxidizing bacteria are self-transmissible or nonself-transmissible, but our results showed that the transmissibility of Hox plasmid pHG21-a varied with the host.

Characterization of recombinant cosmids containing H 2-oxidation genes from Alcaligenes hydrogenophilus

Ten Pseudomonas oxalaticus OX1 harboring recombinant cosmids containing H 2-oxidation genes (hox) from Alcaligenes hydrogenophilus showed H 2-dependent autotrophic growth with different growth rates. Some strains grew autotrophically almost as fast as the strain containing the Hox plasmid pHG21-a. Ten strains had high activity of membrane-bound hydrogenase (MH), but the activity of soluble hydrogenase could not be detected. Sizes of DNA inserts in recombinant cosmids isolated from 10 strains were 26 to 29 kilobases (Kb), and there was a 22-Kb overlapped region. It is suggested that the DNA fragment required for conferring the ability of H 2-dependent autotrophic growth on CO 2-fixing P. oxalaticus appears to span a region of more than 20 Kb in A. hydrogenophilus. Structural genes of MH were found in the right end of the overlapped region by DNA hybridization analysis using oligonucleotide probes corresponding to the conserved sequences of MH genes in Bradyrhizobium japonicum and Rhodobacter capsulatus. The MH was induced by H 2 and repressed by the addition of fructose, as in A. hydrogenophilus. Therefore, the inserted DNA fragment contained the DNA region which determines the regulatory properties as well as structural genes of MH.

Conjugal transfer of hydrogen-oxidizing ability of [formula omitted] to [formula omitted

Conjugal transfer of hydrogen-oxidizing ability (Hox) of the hydrogen bacterium Alcaligenes hydrogenophilus was examined. Intraspecific cross of plasmid pHG21-a that encodes hydrogenases that mediate hydrogen oxidation was most frequent at 25 C; the optimal temperature for growth was 30 C. The plasmid could be transferred from A. hydrogenophilus to Pseudomonas oxalaticus OX1 and OX4, and the resulting strains gained the capacity for autotrophic growth with H 2 and CO 2. Plasmid pHG21-a was maintained in P. oxalaticus OX1 and OX4 as stably as in A. hydrogenophilus .

Isolation of hydrogen-oxidation gene from Alcaligenes hydrogenophilus and its expression in Pseudomonas oxalaticus.

A gene bank of a megaplasmid encoding the hydrogen-oxidizing enzyme system (Hox) in Alcaligenes hydrogenophilus was constructed using a broad host range cosmid vector pVK102, and established in Escherichia coli. Hybrid cosmids containing hox genes were identified by transferring the bank into Pseudomonas oxalaticus OX1 and screening colonies for the ability of H2-dependent autotrophic growth. About 800 colonies were formed under autotrophic conditions. One of the Hox+ transconjugants was isolated and its hydrogenases activities were measured. Although soluble hydrogenase was not detected, the Hox+ transconjugant had four times the membrane-bound hydrogenase activity of A. hydrogenophilus.

Regulation of Autotrophic and Heterotrophic Metabolism in Pseudomonas oxalaticus OX1. Growth on Fructose and on Mixtures of Fructose and Formate in Batch and Continuous Cultures

In Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic CO2 fixation via the Calvin cycle is regulated by repression/derepression. During growth of the organism on fructose alone, the synthesis of ribulosebisphosphate carboxylase (RuBPCase) remained fully repressed, both in batch culture and in fructose-limited continuous cultures at various dilution rates. Growth in batch culture on a mixture of fructose and formate resulted in the simultaneous utilization of both substrates. Under these conditions we observed synthesis of RuBPCase up to high levels, indicating that formate did not merely function as an ancillary energy source in the metabolism of fructose, but stimulated autotrophic CO2 fixation via the Calvin cycle. In subsequent experiments growth of P. oxalaticus on mixtures of fructose and formate was studied in carbon source-limited continuous cultures. In these experiments further evidence was obtained that fructose is a poor source of (co-)repressor molecules for the synthesis of RuBPCase in the presence of formate. Thus, addition of formate to the medium reservoir of a fructose-limited continuous culture resulted in derepression of RuBPCase synthesis at (relatively) high ratios of fructose over formate. In the reverse experiment the specific activity of RuBPCase decreased with increasing concentrations of fructose in the medium reservoir. However, it can be calculated that the total capacity of RuBPCase in the culture to fix CO2 remained constant. In these experiments the dry weight produced on the various mixtures equalled the sum of the dry weight values obtained during growth on the same amounts of the two substrates separately. This indicated that, once RuBPCase was present, autotrophic and heterotrophic carbon assimilation pathways functioned simultaneously and independently of each other. Possible explanations for the low repressing effect of fructose on autotrophic CO2 fixation in P. oxalaticus are discussed.

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.

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

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

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 (SR=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.

Active transport of oxalate by Pseudomonas oxalaticus OX1

Membrane vesicles isolated from oxalate-grown 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-phenazine-methosulphate. 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 (Km = 11 micron) 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.

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 maxATP ] 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 maxATP ]theor. for oxalate was 9.4. True Y maxATP 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.

Substrate inhibition in Pseudomonas 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 50 mM 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 20 mM, whereas oxalate inhibited growth at concentrations above 15 mM. Complete inhibition was not observed even at concentrations of 100 mM. 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 50 mM, indicating that different mechanisms may underlie the observed growth inhibition by the two substrates.

THE FLUORIMETRIC MICRODETERMINATION OF GLYOXYLIC ACID IN BLOOD, URINE AND BACTERIAL EXTRACTS

1. A spectrophotofluorimetric method for the determination of glyoxylic acid in biological materials is described. 2. The method is based on the reaction between glyoxylic acid and resorcinol in acid solution, a fluorescent complex being obtained on the subsequent addition of alkali. 3. The reaction was found to be sensitive and highly specific, the minimum detectable amount of glyoxylic acid being 1.35x10(-8) mole. 4. The urinary excretion of glyoxylic acid by ten normal adults ranged from 1.4 to 4.7mg./24hr. Small but measurable amounts of glyoxylic acid were found in cell-free extracts of Pseudomonas oxalaticus OX1 grown on oxalic acid as a source of carbon. No glyoxylic acid was detected in human serum.