Obligate Thymidine Auxotrophs of Pseudomonas acidovorans

The aminopterin technique was adapted for the isolation of thymidine auxotrophs of Pseudomonas acidovorans. All the mutants isolated responded to thymidine but not to thymine.     

Oxidation of alpha-methylstyrene by Pseudomonas cultures

The work was aimed at studying alpha-methylstyrene oxidation by three active Pseudomonas cultures. P. acidovorans 9 oxidized alpha-methylstyrene only to acetophenone whereas two P. aeruginosa cultures assimilated the compound entirely. Analysis of the intermediate products and enzymes catalyzing the aromatic ring cleavage suggests that these strains oxidize alpha-methylstyrene via a new unknown pathway.     

Thymidine salvage in Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous expression of Escherichia coli thymidine kinase gene.

Unlike enteric bacteria, Pseudomonas spp. generally lack thymidine phosphorylase and thymidine kinase activities, thus preventing their utilization of exogenous thymine or thymidine and precluding specific radioactive labeling of their DNA in vivo. To overcome this limitation, a DNA fragment encoding thymidine kinase (EC 2.7.1.21) from Escherichia coli was cloned into pKT230, a small, broad-host-range plasmid derived from plasmid RSF1010. From transformed E. coli colonies, the recombinant plasmid bearing the thymidine kinase gene was conjugally transferred to Pseudomonas stutzeri, Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes. Thymidine kinase activity was expressed in all of these species, and all gained the ability to incorporate exogenous [2-14C]thymidine into their DNA. Thymidine incorporation into P. stutzeri was enhanced 12-fold more in mutants lacking thymidylate synthetase activity. These mutants produced higher levels of thymidine kinase and were thymidine auxotrophs; thymineless death resulted from removal of thymidine from a growing culture.     

Metabolism of carbohydrate derivatives by Pseudomonas acidovorans.

Wild-type Pseudomonas acidovorans strain A1 was unable to grow on glycerol or glucose as sole source of carbon and energy although it grew well on gluconate. Spontaneous glycerol-positive mutants, which apparently had become permeable to glycerol, were readily isolated, but glucose-positive mutants did not occur. P. acidovorans lacked glucose dehydrogenase and glucokinase, which were sufficient to account for its inability to grow on glucose. Gluconate was degraded exclusively via a noncoordinately induced Entner-Doudoroff pathway. Phosphogluconate dehydrogenase was undetectable. In contrast to P. aeruginosa, P. acidovorans possessed a single glyceraldehyde-phosphate dehydrogenase activity, which was NAD+ specific and constitutive, and an inducible pyruvate kinase. Moreover, growth of glycerol-positive strain K2 on glycerol did not induce any of the enzymes related to metabolism of hexosephosphate derivatives as occurs in fluorescent pseudomonads.     

Incorporation of thymidine into the DNA of actinomycetes. I. Incorporation of exogenous thymidine into the DNA of Thermoactinomyces vulgaris]

The incorporation of exogenous thymidine and thymine into acid-insoluble material of Thermoactinomyces vulgaris has been studied during germination and subsequent growth. Thymine is not incorporated. The incorporation of thymidine stops after a short time due to the rapid breakdown of thymidine to thymine and deoxyribose-1-phosphate by the inducible thymidine phosphorylase. Deoxyadenosine enhances the incorporation of thymidine as well as of thymine and prolongs the tine of uptake. Uridine stimulates only the incorporation of thymidine but not of thymine. These effects can be explained by the function of these substances within the salvage pathway. Deoxyadenosine acts as donor of deoxyribosyl groups being necessary for the conversion of thymine to thymidine by thymidine phosphorylase and uridine inhibits thymidine phosphorylase, and thereby it prevents the degradation of thymidine to thymine. Thymidine is incorporated into alkali-, RNase-and protease-stable, hot TCA-soluble and DNase-sensitive material. That means that the cellular DNA of T. vulgaris can be specifically labelled by radioactive thymidine in the presence of deoxyadenosine and uridine, respectively.     

Pyrimidine catabolism in Pseudomonas aeruginosa

Pyrimidine catabolism in Pseudomonas aeruginosa was investigated. It was found that the pyrimidine bases uracil and thymidine as well as their respective reductive catabolic products could be utilized as sole sources of nitrogen. Reductive degradation of the pyrimidine bases was noted. The reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl-beta-alanine amidohydrolase were all detected in minimal medium grown cells. Induction of pyrimidine catabolism by uracil was observed in this pseudomonad. Pyrimidine degradation in P. aeruginosa was not subject to catabolite repression.     

Construction of a Novel Polychlorinated Biphenyl-Degrading Bacterium: Utilization of 3,4'-Dichlorobiphenyl by Pseudomonas acidovorans M3GY

Pseudomonas acidovorans M3GY is a recombinant bacterium with the novel capacity to utilize a biphenyl congener chlorinated on both rings, 3,4'-dichlorobiphenyl (3,4'-DCBP), as a sole carbon and energy source. Strain M3GY was constructed with a continuous amalgamated culture apparatus (L. Kr?ckel and D. D. Focht, Appl. Environ. Microbiol. 53:2470-2475, 1987) with P. acidovorans CC1(19), a chloroacetate and biphenyl degrader, and Pseudomonas sp. strain CB15(1), a biphenyl and 3-chlorobenzoate degrader. Genetic and phenotypic data showed the recipient parental strain to be P. acidovorans CC1 and the donor parental strain to be Pseudomonas sp. strain CB15. In growth experiments with 3,4'-DCBP as a sole source of carbon, cultures of strain M3GY increased in absorbance from 0.07 to 0.39 in 29 days while reaching a protein concentration of 58 mug ml and 67% substrate dehalogenation. 4-Chlorobenzoate was identified from culture supernatants of strain M3GY by gas chromatography-infrared spectrometry-mass spectrometry; this would be consistent with the oxidation of the m-chlorinated ring through the standard biphenyl pathway. 4-Chlorobenzoate was converted to 4-chlorocatechol, which was metabolized through the meta-fission pathway. The construction of P. acidovorans M3GY, with the novel capability to utilize 3,4'-DCBP, thus involves the complete use of meta-fission pathways for sequential rupture of the biphenyl and chlorobenzoate rings.     

Comparative Immunological Studies of Two Pseudomonas Enzymes

Crystalline preparations of muconate lactonizing enzyme and muconolactone isomerase, two inducible enzymes that catalyze successive steps in the catechol branch of the beta-ketoadipate pathway, were used to prepare antisera. Both enzymes were isolated from a strain of Pseudomonas putida biotype A. The antisera did not cross-react with enzymes of the same bacterial strain that catalyze the chemically analogous steps in the protocatechuate branch of the beta-ketoadipate pathway, carboxymuconate lactonizing enzyme and carboxymuconolactone decarboxylase. The antisera gave heterologous cross-reactions of varying intensities with the muconate lactonizing enzymes and muconolactone isomerases of P. putida biotype B, P. aeruginosa, P. stutzeri, and all biotypes of P. fluorescens, but did not cross-react with the isofunctional enzymes of P. acidovorans, of P. multivorans, and of two bacterial species that belong to other genera. The evolutionary and taxonomic implications of the findings are discussed.     

Incorporation of deoxyribonucleosides into DNA of coryneform bacteria and the relevance of deoxyribonucleoside kinases

In order to obtain basic knowledge of the salvage pathways for DNA synthesis, the ability of Brevibacterium ammoniagenes ATCC 6872 and Micrococcus luteus ATCC 15932 for incorporation of nucleobases and nucleosides was investigated. Only adenine and uracil are incorporated by B. ammoniagenes, whereas M. luteus additionally can utilize deoxyadenosine and, less efficiently, thymidine. In M. luteus, the demonstration of deoxyadenosine kinase and thymidine kinase explains the incorporation data. Uptake of thymidine is of short duration because of rapid breakdown of exogenously supplied thymidine to thymine. At a 540-fold excess pyrimidine deoxyribonucleosides inhibit 14C incorporation from thymidine nearly totally and purine deoxyribonucleosides cut by half the uptake rate, probably by interfering with transport of thymidine. However, as no cessation of thymidine incorporation occurs at these concentrations of purine deoxyribonucleosides, incorporation is finally enhanced. During the initial period of this reduced uptake considerable protection of thymidine from breakdown to thymine is provided by deoxyguanosine, but not by deoxyadenosine. At a 108-fold excess there is actually no inhibition of thymidine uptake by deoxyguanosine and only an insignificant impairment by deoxyadenosine resulting in an ultimate enhancement of 14C incorporation up to 20% of the exogenously supplied thymidine. As there is no salvage pathway for thymidine in B. ammoniagenes due to the absence of thymidine kinase, labelling with adenine and hydrolyzing of the 'contaminated' RNA fraction with 1 M KOH is recommended for measurements of overall DNA synthesis in this strain.     

Genes of the thymidine salvage pathway: thymine-7-hydroxylase from a Rhodotorula glutinis cDNA library and iso-orotate decarboxylase from Neurospora crassa

Genes for two enzymes in the thymidine salvage pathway, thymine-7-hydroxylase (THase; official name thymine dioxygenase) and iso-orotate decarboxylase (IDCase) have been isolated from fungal sources. THase was isolated from a Rhodotorula glutinis cDNA library using a degenerate oligonucleotide based on the published amino acid sequence. The coding sequence was transferred to an Escherichia coli expression system, from which recombinant THase activity was measured using 14C-labeled thymine. The THase sequence shows an almost complete avoidance of codons ending in A or T: 95.8% GC content is present in the third position of codons. A connection between this codon bias and the role of the thymidine salvage pathway in pyrimidine metabolism is proposed. The THase sequence is similar to Group I Fe+2-dependent, alphaKG-dependent dioxygenases. The R. glutinis THase gene was used to locate the probable THase genes in the sequenced genomes of Neurospora crassa and Aspergillus nidulans. The genes neighboring THase in these two genomes are similar to each other, and are similar to the mammalian 2-amino-3-carboxymuconate-6-semialdhyde decarboxylase (ACMSD), leading to their identification as IDCase genes. The N. crassa version was isolated by PCR of genomic DNA, and IDCase activity was measured in recombinant E. coli carrying this gene. A new family of decarboxylases, using similar substrates, is identified by virtue of the protein sequence similarity.     

Regulation of the synthesis of 3-deoxy-D-arabinoheptuloso-7-phosphate-synthase in Pseudomonas bacteria]

Regulation of synthesis of 3-deoxy-D-arabinoheptulose-7-phosphate-synthase (DAHP-synthase) was analysed in 12 strains of Pseudomonas belonging to 10 species. Repression of synthesis of DAHP-synthase was registered in half of strains under study, this being controlled by tyrosin and phenilalanine in P. aeruginosa PAO1 and PAT 2152, P. fluorescens BKMB 896, P. acidovorans BKMB 1251 and P. testosteroni BKMB 1241 and by only phenilalanine in P. maltophilia BKMB 30. In the rest of cases (in P. putida AC29, P. stutzeri BKMB 148, P. mendocina BKMB 1299, P. marginata BKMB 1298, P. vesicularis BKMB 974 and P. fluorescens BKMB 35) the enzyme was synthesized constitutively.     

High-level expression and characterization of a highly functional Comamonas acidovorans xanthine dehydrogenase in Pseudomonas aeruginosa

An improved procedure is described for the high-level expression of Comamonas acidovorans XDH in Pseudomonas aeruginosa PAO1-LAC. The level of functional expression (56 mg protein/L culture) is found to be 7-fold higher than that observed in Escherichia coli and 30-fold higher than that induced in C. acidovorans. Co-expression of the xdhC gene is required for maximal level of functional expression. Comparison of purified preparations of XDH expressed in the absence of xdhC (XDH(AB)) with that expressed in its presence (XDH(ABC)) shows the increased level of activity due to the level of Mo incorporation. The Fe and FAD contents of expressed enzymes are independent of xdhC co-expression. Electron paramagnetic resonance spectroscopy, circular dichroism spectroscopy, metal analysis, and kinetic properties of recombinant purified XDH(ABC) are identical with those exhibited by the native enzyme. This expression system should serve as a valuable tool for further biophysical and mechanistic investigations of xanthine dehydrogenase by site-directed mutagenesis. A method is also described to evaluate the suitability of P. aeruginosa and other organisms as potential expression hosts for five different sources of xdh genes.     

New Gas Chromatographic Characterization Procedure: Preliminary Studies on Some Pseudomonas Species

Strains of saccharolytic and nonsaccharolytic Pseudomonas species were examined by a new single-step gas chromatographic characterization procedure. Cells were digested in a methanolic solution of tetramethylammonium hydroxide pentahydrate, and the digestates were subjected to gas-liquid chromatographic analysis. The chromatograms were examined for similarities and differences in their overall patterns. A single component was defined for use as an internal qualitative and quantitative standardizing component in order to develop relative retention time-relative peak height profiles of each organism. Comparison of these profiles enabled the characterization of strains of Pseudomonas aeruginosa, P. putida, P. cepacia, P. pseudomallei, P. stutzeri, P. pseudoalcaligenes, P. alcaligenes, P. diminuta, P. denitrificans, and P. acidovorans. The P. maltophilia and P. putrefaciens digestates showed chromatograms which were superficially similar yet easily distinguished as belonging to different species. The chromatograms of these two organisms were very different from those of other pseudomonads.     

Genetic and molecular characterization of the Pseudomonas plasmid pVS1

A restriction map of the 30-kb nonconjugative Pseudomonas plasmid pVS1 was constructed. Derivatives of pVS1 obtained in vitro by successive deletions were used to localize on the physical map the determinant for resistance to mercuric ions (carried by transposon Tn501), the gene(s) encoding sulfonamide resistance, a 1.6-kb region affecting plasmid stability and establishment in P. fluorescens ATCC 13525, and a segment required for mobilization of pVS1 by plasmid RP1. The sulfonamide resistance determinant of pVS1 appeared to be closely related to that of transposon Tn21. A mini-pVS1 replicon, pME259, consisting of an essential 1.55-kb segment (designated rep and thought to carry the origin of replication) and a mercury resistance determinant was able to replicate P. aeruginosa PAO but selective pressure was needed for plasmid maintenance. The copy number of pVS1 derivatives was estimated to be 6-8 per chromosome equivalent. Plasmids possessing the essential rep segment plus the adjacent stability region could be established in strains of P. aeruginosa, P. putida, P. fluorescens, P. acidovorans, P. cepacia, P. mendocina, P. stutzeri, P. syringae, Agrobacterium tumefaciens, and Rhizobium leguminosarum.     

Biochemical genetics of tryptophan synthesis in Pseudomonas acidovorans.

Sixty independent tryptophan auxotrophs of Pseudomonas acidovorans were isolated and characterized for nutritional response to intermediates of the pathway, accumulation of intermediates, and levels of tryptophan-synthetic enzymes. Mutants for each of the seven proteins catalyzing the five steps of tryptophan synthesis were obtained. Transductional analysis established three unlinked chromosomal regions: trpE, trpGDC, and trpFBA. The order of the genes within the two clusters was not determined. The levels and enzymatic activities of wild-type and mutant strains indicated that trpE and trpGDC were repressed by tryptophan. In contrast, trpFBA was not derepressed significantly by starvation for tryptophan. The trpG mutants had an additional requirement for p-aminobenzoate, which suggested that anthranilate synthase subunit II also served as glutamine-binding protein in the analogous reaction catalyzed by p-aminobenzoate synthase. In addition, trpD mutants revealed the ability of P. acidovorans to degrade anthranilate via the beta-ketoadipate pathway.     

Regulation of pyrimidine salvage in Aspergillus nidulans: a role for the major regulatory gene are A mediating nitrogen metabolite repression

Summary The synthesis of thymine 7-hydroxylase, an -ketoglutarate dependent dioxygenase, is subject both to nitrogen metabolite repression and to oxygen repression, while synthesis of the other pyrimidine salvage pathway dioxygenase, pyrimidine deoxyribonucleoside 2-hydroxylase, is subject to neither. areA300, an allele of the positive acting regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans, considerably elevates levels of thymine 7-hydroxylase, probably alleviating at least partly both nitrogen metabolite repression and oxygen repression. areA300 has little or no effect on levels of pyrimidine deoxyribonucleoside 2-hydroxylase but does elevate net uptake capacities for thymine, thymidine and deoxyuridine two-fold. areA300 was selected as allowing thymine to supplement a pyrimidine auxotrophy and was found to allow supplementation by thymidine, other pyrimidine nucleosides and pyrimidine salvage intermediates as well. This is the first reported evidence for areA control over an activity(-ies) not directly concerned with nitrogen source utilization.     

The coenzyme A-dependent, non-β-oxidation pathway and not direct deacetylation is the major route for ferulic acid degradation in Delftia acidovorans

The gene loci fcs and ech, encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively, are involved in the ferulic acid catabolism in Delftia acidovorans. The amino acid sequence deduced from ech exhibited 51% identity to the enoyl-CoA hydratase/aldolase from Pseudomonas sp. strain HR199, indicating that the enzyme from D. acidovorans represents a new lineage of this protein. The genes fcs and ech were expressed in Escherichia coli enabling the recombinant strain to transform ferulic acid to vanillin as revealed by photometric and HPLC analysis. An fcs deficient mutant of D. acidovorans was unable to grow on ferulic acid. The obtained data suggest that in contrast to a previous publication the biotechnologically interesting direct non-oxidative deacetylation mechanism of ferulic acid cleavage is not realized in D. acidovorans. Instead, ferulic acid degradation in D. acidovorans proceeds via a coenzyme A-dependent non-beta-oxidative pathway.     

Studies on the utilization of dietary thymidine and thymine by Drosophila melanogaster larvae

Deoxycytidine improves tolerance of Drosophila melanogaster to thymidine block, suggesting the presence of deoxycytidine kinase. At appropriate concentrations, a mixture of thymidine and deoxycytidine allows larvae to tolerate a higher concentration of 5-fluoro-2′-deoxyuridine than is tolerated with either thymidine or deoxycytidine alone. Thus, at this high concentration, 5-fluoro-2′-deoxyuridine appears to act primarily upon thymidylate synthetase, as it does at lower concentrations, rather than upon RNA metabolism, as has been suggested previously. Larvae can also be rescued from 5-fluoro-2′-deoxyuridine-induced death by a high concentration of thymine. The effect is enhanced by the presence of deoxyadenosine. Since this compound is known to increase the intracellular concentration of deoxyribose-1-phosphate, the main effect of thymine is probably due to its salvage utilization as a thymidine source, via the anabolic functioning of thymidine phosophorylase.     

Mineralization of paraoxon and its use as a sole C and P source by a rationally designed catabolic pathway in Pseudomonas putida

Organophosphate compounds, which are widely used as pesticides and chemical warfare agents, are cholinesterase inhibitors. These synthetic compounds are resistant to natural degradation and threaten the environment. We constructed a strain of Pseudomonas putida that can efficiently degrade a model organophosphate, paraoxon, and use it as a carbon, energy, and phosphorus source. This strain was engineered with the pnp operon from Pseudomonas sp. strain ENV2030, which encodes enzymes that transform p-nitrophenol into beta-ketoadipate, and with a synthetic operon encoding an organophosphate hydrolase (encoded by opd) from Flavobacterium sp. strain ATCC 27551, a phosphodiesterase (encoded by pde) from Delftia acidovorans, and an alkaline phosphatase (encoded by phoA) from Pseudomonas aeruginosa HN854 under control of a constitutive promoter. The engineered strain can efficiently mineralize up to 1 mM (275 mg/liter) paraoxon within 48 h, using paraoxon as the sole carbon and phosphorus source and an inoculum optical density at 600 nm of 0.03. Because the organism can utilize paraoxon as a sole carbon, energy, and phosphorus source and because one of the intermediates in the pathway (p-nitrophenol) is toxic at high concentrations, there is no need for selection pressure to maintain the heterologous pathway.