Degradation of pyrimidine ribonucleosides by Pseudomonas aeruginosa

Pyrimidine ribonucleoside degradation in the human pathogen Pseudomonas aeruginosa ATCC 15692 was investigated. Either uracil, cytosine, 5-methylcytosine, thymine, uridine or cytidine supported P. aeruginosa growth as a nitrogen source when glucose served as the carbon source. Using thin-layer chromatographic analysis, the enzymes nucleoside hydrolase and cytosine deaninase were shown to be active in ATCC 15692. Compared to (NH₄)₂SO₄-grown cells, nucleoside hydrolase activity in ATCC 15692 approximately doubled after growth on 5-methylcytosine as a nitrogen source while its cytosine deaminase activity increased several-fold after growth on the pyrimidine bases and ribonucleosides examined as nitrogen sources. Regulation at the level of protein synthesis by 5-methylcytosine was indicated for nucleoside hydrolase and cytosine deaminase in P. aeruginosa.     

Pyrimidine base and ribonucleoside utilization by thePseudomonas alcaligenes group

Pyrimidine base and ribonucleoside utilization was investigated in the two type strains of thePseudomonas alcaligenes group. As sole sources of nitrogen, the pyrimidine bases uracil, thymine and cytosine as well as the dihydropyrimidine bases dihydrouracil and dihydrothymine supported the growth ofPseudomonas pseudoalcaligenes ATCC 17440 but neither these bases nor pyrimidine nucleosides supportedPseudomonas alcaligenes ATCC 14909 growth. Ribose, deoxyribose, pyrimidine and dihydropyrimidine bases as well as pyrimidine nucleosides failed to be utilized by eitherP. pseudoalcaligenes orP. alcaligenes as sole carbon sources. The activities of the pyrimidine salvage enzymes nucleoside hydrolase, cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase were detected in cell-free extracts ofP. pseudoalcaligenes andP. alcaligenes. InP. pseudoalcaligenes, the levels of cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase could be affected by the nitrogen source present in the culture medium.     

Importance of codB for new codA-based markerless gene deletion in Gluconobacter strains

For the detailed molecular analysis, genomic modification, and application of acetic acid bacteria such as Gluconobacter in biotechnological processes, a simple markerless deletion system is essential. The available methods have either low efficiencies or their applicability is restricted to strains containing an upp mutation. We now developed a method based on counterselection by cytosine deaminase, encoded by the codA gene from Escherichia coli, in the presence of the fluorinated pyrimidine analogue 5-fluorocytosine (FC). The codA-encoded enzyme converts nontoxic FC to toxic 5-fluorouracil, which is channeled into the metabolism by the uracil phosphoribosyltransferase, encoded by the chromosomal upp gene of Gluconobacter. We found that the presence of E. coli codB, encoding a cytosine permease, was needed for a high efficiency of gene deletion. The system is applicable in wild-type strains because no preceding deletions are required. Based on the fact that a codA gene is absent and an upp gene is present in almost all acetic acid bacteria sequenced so far, the method should also be applicable for other genera of the Acetobacteraceae.     

Pathways of Pyrimidine Salvage in <Emphasis Type="Italic">Pseudomonas</Emphasis> and Former <Emphasis Type="Italic">Pseudomonas</Emphasis>: Detection of Recycling Enzymes Using High-Performance Liquid Chromatography

Pyrimidine salvage pathways are vital for all bacteria in that they share in the synthesis of RNA with the biosynthetic pathway in pyrimidine prototrophs, while supplying all pyrimidine requirements in pyrimidine auxotrophs. Salvage enzymes that constitute the pyrimidine salvage pathways were studied in 13 members of Pseudomonas and former pseudomonads. Because it has been established that all Pseudomonas lack the enzyme uridine/cytidine kinase (Udk) and all contain uracil phosphoribosyl transferase (Upp), these two enzymes were not included in this experimental work. The enzymes assayed were: cytosine deaminase [Cod: cytosine + H₂O → uracil + NH₃], cytidine deaminase [Cdd: cytidine + H₂O → uridine + NH₃], uridine phosphorylase [Udp: uridine + P_i ↔ uracil + ribose – 1 - P], nucleoside hydrolase [Nuh: purine/pyrimidine nucleoside + H₂O → purine/pyrimidine base + ribose], uridine hydrolase [Udh: uridine/cytidine + H₂O → uracil/cytosine + ribose]. The assay work generated five different Pyrimidine Salvage Groups (PSG) designated PSG1 – PSG5 based on the presence or absence of the five enzymes. These enzymes were assayed using reverse phase high-performance liquid chromatography techniques routinely carried out in our laboratory. Escherichia coli was included as a standard, which contains all seven of the above enzymes.     

Concomitant expression of E. coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5-fluorocytosine

The prodrug activation system formed by the E. coli codA gene encoding cytosine deaminase (CD) and 5-fluorocytosine (5-FC) developed for selective cancer chemotherapy suffers from a sensitivity limitation in many tumour cells. In an attempt to improve the CD/5-FC suicide association, we combined the E. coli upp gene encoding uracil phosphoribosyltransferase (UPRT) with codA gene to create the situation prevailing in E. coli, a bacterium very efficient in metabolising 5-FC. The constitutive expression of the two genes cloned on an E. coli-animal cell shuttle plasmid either in a linked or in a fused configuration was evaluated in E. coli strains selected and engineered to mimic the 5-FC metabolism encountered in mammalian cells. The simultaneous expression of codA and upp genes generated a cooperative effect resulting in a dramatic increase in 5-FC sensitivity of cells compared to the expression of codA alone. Furthermore, it was shown that the association of UPRT with CD facilitated the uptake of 5-FC, in the situation where the drug penetrates cells by passive diffusion as in mammalian cells, by directly channeling 5-fluorouracil, the product of CD, to 5-fluoroUMP, the product of UPRT.     

Pyrimidine ribonucleoside catabolism in Pseudomonas fluorescens biotype A

The pyrimidine ribonucleosides uridine or cytidine were shown to serve as a source of nitrogen or carbon for the growth of Pseudomonas fluorescens strain A126. After incubation of either pyrimidine ribonucleoside with extracts of this strain, the resultant catabolic products were detected by thin-layer chromatography. It was found that pyrimidine ribonucleoside catabolism in this pseudomonad involved the enzymes nucleoside hydrolase and cytosine deaminase. The specific activities of both these enzymes could be influenced by the nitrogen or carbon source present in the medium.     

Cytosine deaminase as a negative selective marker for Arabidopsis

Cytosine deaminase (CD), produced by prokaryotes but not by higher eukaryotes including plants, deaminates cytosine to uracil. The enzyme likewise converts 5-fluorocytosine (5FC), which by itself is not toxic, to 5-fluorouracil (5FU), which is toxic. The Escherichia coli codA-coding sequence encoding CD, together with appropriate regulatory elements, was introduced into Arabidopsis. Neither untransformed controls, nor transgenic plants expressing no CD mRNA, were sensitive to 5FC. Conversely, for most transgenic plants expressing CD mRNA, in the presence of 5FC calli and seedlings failed to proliferate, and seeds failed to germinate. A few transgenic plants with many codA copies expressed less CD mRNA and remained insensitive to 5FC, which likely reflected epigenetic repeat-induced gene silencing. Thus 5FC, presumably through conversion by the enzyme to 5FU, can be used to select against plants that express CD.     

Role of cytosine deaminase and β-alanine-pyruvate transaminase in pyrimidine base catabolism by Burkholderia cepacia

A determination of the possible role of the salvage enzyme cytosine deaminase or -alanine-pyruvate transaminase in the catabolism of the pyrimidine bases uracil and thymine by the opportunistic pathogen Burkholderia cepacia ATCC 25416 was undertaken. It was of interest to learn whether these enzymes were influenced by cell growth on pyrimidine bases and their respective catabolic products to the same degree as the pyrimidine reductive catabolic enzymes were. It was found that cytosine deaminase activity was influenced very little by cell growth on the pyrimidines tested. Using glucose as the carbon source, only B. cepacia growth on 5-methylcytosine as a nitrogen source increased deaminase activity by about three-fold relative to (NH₄)₂SO₄-grown cells. In contrast, the activity of –alanine-pyruvate transaminase was observed to be at least double in glucose-grown ATCC 25416 cells when pyrimidine bases and catabolic products served as nitrogen sources instead of (NH₄)₂SO₄. Transaminase activity in the B. cepacia glucose-grown cells was maximal after the strain was grown on either uracil or 5-methylcytosine as a nitrogen source compared to (NH₄)₂SO₄-grown cells. A possible role for -alanine-pyruvate transaminase in pyrimidine base catabolism by B. cepacia would seem to be suggested from the similarity in how its enzyme activity responded to cell growth on pyrimidine bases and catabolic products when compared to the response of the three reductive catabolic enzymes.     

Cytosine deaminase as a substrate-dependent negative selectable marker in Brassica napus

The enzyme cytosine deaminase, encoded by the codA gene, catalyzes the deamination of the non- toxic compound 5-fluorocytosine (5-FC) to the highly toxic compound 5-fluorouracil (5-FU). Cytosine deaminase activity is not found in higher plants and Brassica napus seedlings are unaffected by the presence of 5-FC in the growth medium. In codA-transformed B. napus seedlings, expression of cytosine deaminase results in a reduction of root and hypocotyl lengths, and a severe suppression of true leaf development. This phenotype is dependent on the presence of the 5-FC substrate and no effects are seen in plants grown in the absence of the substrate or in sibling plants lacking the transgene. The codA transformants have been assessed over three generations of growth and in each generation the transgene is stably inherited and confers the same 5-FC-sensitive phenotype. Transfer of 5-FC-sensitive seedlings to soil results in the restoration of normal growth in up to 100% of the seedlings. These results indicate that codA is a versatile dominant marker gene that can be used effectively in B. napus for substrate-dependent negative selection. Received: 24 June 1999 / Accepted: 22 July 1999     

Degradation of pyrimidine ribonucleosides by Pseudomonas aeruginosa

Pyrimidine ribonucleoside degradation in the human pathogen Pseudomonas aeruginosa ATCC 15692 was investigated. Either uracil, cytosine, 5-methylcytosine, thymine, uridine or cytidine supported P. aeruginosa growth as a nitrogen source when glucose served as the carbon source. Using thin-layer chromatographic analysis, the enzymes nucleoside hydrolase and cytosine deaninase were shown to be active in ATCC 15692. Compared to (NH₄)₂SO₄-grown cells, nucleoside hydrolase activity in ATCC 15692 approximately doubled after growth on 5-methylcytosine as a nitrogen source while its cytosine deaminase activity increased several-fold after growth on the pyrimidine bases and ribonucleosides examined as nitrogen sources. Regulation at the level of protein synthesis by 5-methylcytosine was indicated for nucleoside hydrolase and cytosine deaminase in P. aeruginosa.     

Osmoregulation in the model organismEscherichia coli: genes governing the synthesis of glycine betaine and trehalose and their use in metabolic engineering of stress tolerance

Glycine betaine is known to be the preferred osmoprotectant in many bacteria, and glycine betaine accumulation has also been correlated with increased cold tolerance. Trehalose is often a minor osmoprotectant in bacteria and it is a major determinant for desiccation tolerance in many so-called anhydrobiotic organisms such as baker's yeast(Saccharomyces cerevisiae). Escherichia coli has two pathways for synthesis of these protective molecules; i.e., a two-step conversion of UDP-glucose and glucose-6-phosphate to trehalose and a two-step oxidation of externally-supplied choline to glycine betaine. The genes governing the choline-to-glycine betaine pathway have been studied inE. coli and several other bacteria and higher plants. The genes governing UDP-glucose-dependent trehalose synthesis have been studied inE. coli andS. cerevisiae. Because of their well-documented function in stress protection, glycine betaine and trehalose have been identified as targets for metabolic engineering of stress tolerance. Examples of this experimental approach include the expression of theE. coli betA andArthrobacter globiformis codA genes for glycine betaine synthesis in plants and distantly related bacteria, and the expression of theE. coli otsA and yeastTPS1 genes for trehalose synthesis in plants. The published data show that glycine betaine synthesis protects transgenic plants and phototrophic bacteria against stress caused by salt and cold. Trehalose synthesis has been reported to confer increased drought tolerance in transgenic plants, but it causes negative side effects which is of concern. Thus, the much-used model organismE. coli has now become a gene resource for metabolic engineering of stress tolerance.     

Growth Inhibition by Purine Derivatives and Its Reversal by Pyrimidine Derivatives in a Mutant of Escherichia coli K12

An adenosine-sensitive mutant was isolated from Escherichia coli K12 derivative strain C600. This mutant (designated as PS100) grew slower than parental strain C600in a minimal medium, and its growth was completely inhibited by addition of all kinds of purine bases, nucleosides and nucleotides tested. On the other hand, this growth inhibitory effect of purine derivatives was reversed by co-addition of uridine to the medium. Other pyrimidine derivatives such as uracil, UMP,cytosine, cytidine, CMP and thymidine were also effective for this reversal. The mutant strain, PS100, showed a lower level (7%) of activity for orotate phosphoribosyltransferase than strain C600 did, and accumulated orotic acid in the growth medium. Lysogenization of strain PS100 with λ transducing phage containing the gene for orotate phosphoribosyltransferase (pyrE) resulted in restoration of the activity for orotate phosphoribosyltransferase and removal of growth inhibition by purine derivatives.     

Improved cytotoxic effects of Salmonella‐producing cytosine deaminase in tumour cells

In order to increase the cytotoxic activity of a Salmonella strain carrying a salicylate‐inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the Escherichia coli codA gene cloned under the control of the Pm promoter have been improved by using the T7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG. Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5‐fluorocytosine, a 5‐fluorouracyl resistant Salmonella strain has been constructed by deleting its upp gene sequence. This new Salmonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic Salmonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different Salmonella strains in tumour cell cultures.     

A co-transformation system to produce transgenic grapevines free of marker genes

A co-transformation system was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. ‘Thompson Seedless’ somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bi-directional dual promoter complex. Our technique included a short positive selection phase on medium containing 100 mg l⁻¹ kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg l⁻¹ 5-fluorocytosine. We regenerated 25 stable EGFP expressing transgenic lines. PCR analysis confirmed 18 lines contained only the egfp gene, whereas the remaining contained both egfp and codA/nptII genes. Presumably, the 18 monogenic lines arose through cross protection by being in close proximity to cells that expressed nptII and thus detoxified kanamycin in the immediate vicinity. This is the first report for grapevine using a combination of positive and negative selection to produce transgenic plants that do not contain marker genes.     

Control of the pyrimidine biosynthetic pathway in Pseudomonas pseudoalcaligenes

The five de novo enzyme activities unique to the pyrimidine biosynthetic pathway were found to be present in Pseudomonas pseudoalcaligenes ATCC 17440. A mutant strain with 31-fold reduced orotate phosphoribosyltransferase (encoded by pyrE) activity was isolated that exhibited a pyrimidine requirement for uracil or cytosine. Uptake of the nucleosides uridine or cytidine by wild-type or mutant cells was not detectable; explaining the inability of the mutant strain to utilize either nucleoside to satisfy its pyrimidine requirement. When the wildtype strain was grown in the presence of uracil, the activities of the five de novo enzymes were depressed. Pyrimidine limitation of the mutant strain led to the increase in aspartate transcarbamoylase and dihydroorotate dehydrogenase activities by more than 3-fold, and dihydroorotase and orotidine 5-monophosphate decarboxylase activities about 1.5-fold, as compared to growth with excess uracil. It appeared that the syntheses of the de novo enzymes were regulated by pyrimidines. In vitro regulation of aspartate transcarbamoylase activity in P. pseudoalcaligenes ATCC 17440 was investigated using saturating substrate concentrations; transcarbamoylase activity was inhibited by P_i, PP_i, uridine ribonucleotides, ADP, ATP, GDP, GTP, CDP, and CTP.     

Translocation of a discrete piece of deoxyribonucleic acid carrying an amp gene between replicons in Eschericha coli.

Uptake and intracellular transformation of pyrimidines supplying cells of the yeast Rhodotorula glutinis with nitrogen have been studied. The amine nitrogen of cytosine was found to be the easiest to utilize. The presence in the medium of inorganic ammonia along with cytosine had a slight effect on cytosine deaminase (EC 3.5.4.1) activity. The uracil produced entered into the nutrient medium with no fission break of the pyridmidine ring. In the absence of any other source of nitrogen, the cells of the yeast R. glutinis utilized nitrogen of the pyrimidine ring of oxypyrimidines. Catabolism of uracil followed the reductive pattern, with release of carbon dioxide; this was accompanied by synthesis of the key enzyme of pyrimidine catabolism, dihydrouracil dehydrogenase (EC 1.3.1.1), whose activity rose 10-fold. With thymidne as the sole source of nitrogen, the lag-phase growth of the yeast cells was maximum. Catabolism of the pyrimidine ring of thymine was possibly preceded by its transformation into uracil. With no source of nitrogen easily utilized, the uridine 5'-monophosphate content in the generally acid-soluble pool rose. Our discussion of the regulation of catabolism of exogenous pyrimidine bases by the yeast R. glutinis takes into account the fact that transformations of pyrimidine bases are determined by how easily the cells can use a particular base as a source of nitrogen.     

Enhanced germination under high-salt conditions of seeds of transgenicArabidopsis with a bacterial gene (codA) for choline oxidase

Arabidopsis thaliana was transformed previously with thecodA gene from the soil bacteriumArthrobacter globiformis. This gene encodes choline oxidase, the enzyme that converts choline to glycinebetaine. Transformation with thecodA gene significantly enhanced the tolerance of transgenic plants to low temperature and high-salt stress. We report here that seeds of transgenic plants that expressed thecodA gene were also more tolerant to salt stress during germination than seeds of non-transformed wild-type plants. Seedlings of transgenic plants grew more rapidly than those of wild-type plants under salt-stress conditions. Furthermore, exogenously applied glycinebetaine was effective in alleviating the harmful effects of salt stress during germination of seeds and growth of young seedlings, a result that suggests that it was glycinebetaine that had enhanced the tolerance of the transgenic plants. These observations indicate that synthesis of glycinebetaine in transgenic plantsin vivo, as a result of the expression of thecodA gene, might be veryuseful in improving the ability of crop plants to tolerate salt stress. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Regulation of the Pyrimidine Biosynthetic Pathway in <Emphasis Type="Italic">Pseudomonas mucidolens</Emphasis>

Control of pyrimidine biosynthesis was examined in Pseudomonas mucidolens ATCC 4685 and the five de novo pyrimidine biosynthetic enzyme activities unique to this pathway were influenced by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. When uracil was supplemented to glucose-grown ATCC 4685 cells, activities of four de novo enzymes were depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the glucose-grown mutant strain cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase activities to increase by more than 3-fold while OMP decarboxylase activity increased by 2.7-fold. The syntheses of the de novo enzymes appeared to be regulated by pyrimidines. At the level of enzyme activity, aspartate transcarbamoylase activity in P. mucidolens ATCC 4685 was subject to inhibition at saturating substrate concentrations. Transcarbamoylase activity was strongly inhibited by UTP, ADP, ATP, GTP and pyrophosphate.     

The PurR mutation of Drosophila melanogaster confers resistance to purine and 2,6-diaminopurine by elevating adenosine deaminase activity

Summary Media supplemented with purine (7H-imidazo[4,5-d]pyrimidine) or the purine analogue 2,6-diaminopurine (DAP) can be employed to select several classes of purine-resistant variants from mutagenized cultures of Drosophila. One class results in elevated resistance to purine and diaminopurine which is correlated with elevated activity of the enzyme adenosine deaminase (adenosine aminohydrolase=EC 3.5.4.4). The first member of this class, PurR, maps to position 82± in the right arm of the second chromosome. The PurR mutation causes an elevation of adenosine deaminase (ADA) enzyme activity, apparently by altering a thermolabile, ADA-specific repressor. PurR may thus encode a negative regulator of adenosine deaminase activity similar to the ADA-binding protein found in mammalian systems.     

Two-dimensional electrophoretic analysis of the regulation of SOS proteins in three ssb mutants

A previously undescribed mutation in the ssb gene, which codes for a major single strand DNA binding protein essential for DNA repelication, was mapped on the Escherichia coli Chromosome. Three ssb mutants were analyzed under parallel physiological conditions for the induction of SOS proteins (products of recA, uvrA, and an unknown gene), the production of mutants, the induction of lambda prophage, and sensitivity to DNA damaging agents. Two-dimensional electrophoretic techniques were used to quantitate changes in the rate of synthesis of proteins. The previously unpublished position of the uvrA gene-product in the two-dimensional matrix of E. coli proteins was described. These ssb strains exhibited varying sensitivities to ultraviolet irradiation and methylmethane sulfonate that correlated with the rate of constitutive synthesis of SOS proteins, spontaneous commitment to virulent growth of lambda lysogens, and elevation of endogenous mutation rates.Dedicated to the memory of Roger Y. Stanier: to his fascination for diverse microbial lifeforms that catalyzed curiosity in his associates, to his intellectual aura that elicited deep respect, to his pursuit of scientific truth that promoted the highest research ethics, to his friendly nature that encouraged my growth as a scientist and enkindled my love for Roger