Thymidine For Growth Research Articles

Chromosomal integration of heterologous oxalate decarboxylase in Lactobacillus plantarum WCFS1 using mobile genetic element Ll.LtrB.

Lactobacillus plantarum WCFS1 (L. plantarum WCFS1) is commonly used as a potential cell factory because of its 'generally recognized as safe' status. The plasmid instability and the presence of antibiotic selection marker complicate the application of genetically modified L. plantarum in human clinical trials. In the present study, we aimed to integrate oxalate decarboxylase (oxdC) gene of Bacillus subtilis origin by targeted chromosomal mutation in L. plantarum using mobile genetic element Ll.LtrB as a therapeutic tool against calcium oxalate stone disease. oxdC expression cassette was constructed and integrated into a targeted gene, thymidylate synthase (thyA) in the L. plantarum genome. The dependence on external thymidine for growth and survival was established by live dead population assay and SEM (scanning electron microscopy) analysis. The western blotting assay showed the secretion of 44kDa OxdC protein in the culture supernatant of L. plantarum ∆thyA:OxdC. The biologically contained recombinant strain significantly reduced the oxalate concentration by 53% and exhibited a loss of viability when introduced to environmental samples. Biologically contained L. plantarum secreting OxdC constructed using group II intron has the ability to degrade oxalate present in the extracellular environment and could be used as a therapeutic tool for the calcium oxalate stone disease.

Investigation of local probiotic bacteria as live sublingual vaccines for treatment of atopic disease in vivo

Background & Aims: The use of living, genetically modified bacteria is an effective approach for topical delivery of immunomodulatory proteins. This strategy circumvents systemic side effects and allows long-term treatment of chronic diseases. However, treatment of patients with a living, genetically modified bacterium raises questions about the safety for human subjects per se and the biologic containment of the transgene. Methods: We treated Crohn’s disease patients with genetically modified Lactococcus lactis (LL-Thy12) in which the thymidylate synthase gene was replaced with a synthetic sequence encoding mature human interleukin-10. Ten patients were included in a placebo-uncontrolled trial. Patients were assessed daily for the presence of potential adverse effects by direct questioning and assessment of disease activity. We evaluated the presence and kinetics of LL-Thy12 release in the stool of patients by conventional culturing and quantitative polymerase chain reaction of LL-Thy12 gene sequences. Results: Treatment with LL-Thy12 was safe because only minor adverse events were present, and a decrease in disease activity was observed. Moreover, fecally recovered LL-Thy12 bacteria were dependent on thymidine for growth and interleukin-10 production, indicating that the containment strategy was effective. Conclusions: Here we show that the use of genetically modified bacteria for mucosal delivery of proteins is a feasible strategy in human beings. This novel strategy avoids systemic side effects and is biologically contained; therefore it is suitable as maintenance treatment for chronic intestinal disease.

A Phase I Trial With Transgenic Bacteria Expressing Interleukin-10 in Crohn’s Disease

The use of living, genetically modified bacteria is an effective approach for topical delivery of immunomodulatory proteins. This strategy circumvents systemic side effects and allows long-term treatment of chronic diseases. However, treatment of patients with a living, genetically modified bacterium raises questions about the safety for human subjects per se and the biologic containment of the transgene. We treated Crohn's disease patients with genetically modified Lactococcus lactis (LL-Thy12) in which the thymidylate synthase gene was replaced with a synthetic sequence encoding mature human interleukin-10. Ten patients were included in a placebo-uncontrolled trial. Patients were assessed daily for the presence of potential adverse effects by direct questioning and assessment of disease activity. We evaluated the presence and kinetics of LL-Thy12 release in the stool of patients by conventional culturing and quantitative polymerase chain reaction of LL-Thy12 gene sequences. Treatment with LL-Thy12 was safe because only minor adverse events were present, and a decrease in disease activity was observed. Moreover, fecally recovered LL-Thy12 bacteria were dependent on thymidine for growth and interleukin-10 production, indicating that the containment strategy was effective. Here we show that the use of genetically modified bacteria for mucosal delivery of proteins is a feasible strategy in human beings. This novel strategy avoids systemic side effects and is biologically contained; therefore it is suitable as maintenance treatment for chronic intestinal disease.

TheSalmonella entericaSerovar TyphitsxGene, Encoding a Nucleoside-Specific Porin, Is Essential for Prototrophic Growth in the Absence of Nucleosides

The Salmonella enterica serovar Typhi tsx gene encodes a porin that facilitates the import of nucleosides. When serovar Typhi is grown under anaerobic conditions, Tsx is among the outer membrane proteins whose expression increases dramatically. This increase in expression is due, at least in part, to increased transcription and is dependent on Fnr but not on ArcA. A mutant derivative of serovar Typhi strain STH2370 with a deletion of the tsx gene is an auxotroph that requires either adenosine or thymidine for growth on minimal medium. In contrast, an otherwise isogenic nupG nupC double mutant, defective in the inner membrane nucleoside permeases, is a prototroph. Because anaerobic growth enhances the virulence of serovar Typhi in vitro, we assessed the role that the tsx gene plays in pathogenicity and found that the serovar Typhi STH2370 Deltatsx mutant is defective in survival within human macrophage-like U937 cells. To understand why the Deltatsx mutant is an auxotroph, we selected for insertions of minitransposon T-POP in the Deltatsx genetic background that restored prototrophy. One T-POP insertion that suppressed the Deltatsx mutation in the presence of the inducer tetracycline was located upstream of the pyrD gene. The results of reverse transcription-PCR analysis showed that addition of the inducer decreased the rate of pyrD transcription. These results suggest that the Tsx porin and the balance of products of the tsx and pyrD genes play critical roles in membrane assembly and integrity and thus in the virulence of serovar Typhi.

Construction and characterization of a nonproliferative El Tor cholera vaccine candidate derived from strain 638.

In recent clinical assays, our cholera vaccine candidate strain, Vibrio cholerae 638 El Tor Ogawa, was well tolerated and immunogenic in Cuban volunteers. In this work we describe the construction of 638T, a thymidine auxotrophic version of improved environmental biosafety. In so doing, the thyA gene from V. cholerae was cloned, sequenced, mutated in vitro, and used to replace the wild-type allele. Except for its dependence on thymidine for growth in minimal medium, 638T is essentially indistinguishable from 638 in the rate of growth and morphology in complete medium. The two strains showed equivalent phenotypes with regard to motility, expression of the celA marker, colonization capacity in the infant mouse cholera model, and immunogenicity in the adult rabbit cholera model. However, the ability of this new strain to survive environmental starvation was limited with respect to that of 638. Taken together, these results suggest that this live, attenuated, but nonproliferative strain is a new, promising cholera vaccine candidate.

Implantation d'un mutant de Escherichia coli exigeant en acide diaminopimélique dans le tube digestif de souris gnotoxéniques

A DAP- auxotroph mutant of Escherichia coli DP50 requiring DAP and thymidine for growth was used as the receptor strain in genetic engineering. It failed to be implanted in axenic mice. However, when an inoculum containing more than 10(7) bacteria/ml was used, the DAP+ reverse mutant devoid of requirement for DAP became implanted. When axenic mice were previously associated with Clostridium difficile containing DAP in the cell wall, the strain DAP- became implanted even when the inoculum was too small to permit implantation in axenic mice. Conversely, C. butyricum and C. perenne, whose cell walls also contain DAP, did not allow the establishment of a DAP- mutant. In animals associated with complex human flora without enterobacteria, neither of the 2 DAP- and DAP+ mutants became implanted.

Isolation of a dihydrofolate reductase-deficient mutant of Escherichia coli.

A strain of Escherichia coli was isolated in which dihydrofolate reductase was not detected by an enzyme assay or by competition for antibody. This strain requires methionine, glycine, a purine, and thymidine for growth in addition to the auxotrophic requirements of the parent strain. It was found to be useful as a recipient of plasmids harboring dihydrofolate reductase genes.

Thymine inhibits suppression by an Escherichia coli nonsense and frameshift suppressor.

A mutant strain of Escherichia coli suppressed a frameshift and some UAG, UAA, and UGA mutants of bacteriophage T4 at 37 degrees C but not at 31 degrees C. This suppression was inhibited by the addition of thymine or thymidine to the medium used to test phage growth. Furthermore, the suppressor strain required thymine or thymidine for growth on minimal medium at 43 degrees C and if this auxotrophy was removed by reversion or recombination the strain no longer suppressed. These results suggest a link between thymidine nucleotide biosynthesis and suppression.

Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity.

Mutants of Chinese hamster ovary cells lacking dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate:NADP+ oxidoreductase; EC 1.5.1.3) activity were isolated after mutagenesis and exposure to high-specific-activity [3H]deoxyuridine as a selective agent. Fully deficient mutants could not be isolated starting with wild-type cells, but could readily be selected from a putative heterozygote that contains half of the wild-type level of dihydrofolate reductase activity. The heterozygote itself was selected from wild-type cells by using [3H]deoxyuridine together with methotrexate to reduce intracellular dihydrofolate reductase activity. Fully deficient mutants require glycine, a purine, and thymidine for growth; this phenotype is recessive to wild type in cell hybrids. Revertants have been isolated, one of which produces a heat-labile dihydrofolate reductase activity. These mutants may be useful for metabolic studies relating to cancer chemotherapy and for fine-structure genetic mapping of mutations by using available molecular probes for this gene.

Thymidine-requiring mutants of Salmonella typhimurium that are defective in deoxyuridine 5'-phosphate synthesis

In a Salmonella typhimurium strain made diploid for the thy region by introduction of the Escherichia coli episome, F'15, mutants resistant to trimethoprim in the presence of thymidine were selected. One was shown to be defective in deoxyuridine 5'-phosphate (dUMP) synthesis; it requires deoxyuridine or thymidine for growth and is sensitive to trimethoprim in the presence of deoxyuridine. Genetic studies showed that the mutant is mutated in two genes, dcd and dum, located at 70 and 18 min, respectively, on the Salmonella linkage map. The dcd gene cotransduces 95% with udk, the structural gene for uridine kinase. Both mutations are necessary to create a deoxyuridine requirement, providing evidence for the existence of two independent pathways for dUMP synthesis. Pool studies showed that a dum mutation by itself causes a small decrease in the deoxythymidine 5'-triphosphate (dTTP) pool of the cells, whereas a dcd mutation results in a much more marked decrease. The double mutant dcd dum, when incubated in the absence of deoxyuridine, contains barely detectable levels of dTTP. Enzyme analysis revealed that dcd encodes deoxycytidine 5'-triphosphate deaminase. The gene product of the dum gene has not yet been identified; it does not encode either subunit of ribonucleoside diphosphate reductase or deoxyuridine 5'-triphosphate pyrophosphatase. Mutants deleted for the dcd-udk region of the S. typhimurium chromosome were isolated.

Thymine Dependent Strains of Escherichia Coli Selected By Trimethoprim-Sulphamethoxazole Therapy

Six strains of thymine-dependent Escherichia coli were isolated fron the urine of 6 patients receiving trimethoprim-sulphamethoxazole therapy. These organisms require exogenous thymine or thymidine for growth and form long filaments in media deficient in these substances. These mutants can be identified in clinical material by use of a thymidine disc applied to thymidine-deficient media. As these strains are not susceptible to trimethoprim-sulphamethoxazole therapy, this combination should be withdrawn when they are identified.

The Role of Vitamin B12 in Metabolic Processes

Publisher Summary This chapter analyzes the role of vitamin B 12 in metabolic processes. The fundamental source of vitamin B 12 in animal nutrition appears to be in nonphotosynthetic organisms. A microbiological synthesis of vitamin B 12 may take place in the rumen and elsewhere in the digestive tract of cattle, providing a supply of the vitamin that is accumulated in the liver, thus accounting for the effectiveness of extracts of this organ in the treatment of pernicious anemia. The growth of certain thermobacteria is stimulated by thymidine or vitamin B 12 . The requirement for vitamin B 12 in certain lactic acid organisms can be obviated by the addition of ascorbic acid. Microorganisms that respond to vitamin B 12 or thymidine for growth on a medium containing pteroylglutamic acid may be divided into five groups depending on their response to various desoxyribosides, vitamin B 12 and ascorbic acid. Studies also show that vitamin B 12 is concerned in reactions involving a single carbon transfer in the synthesis of methionine, serine, thymine and purines in E. coli . Studies also show that vitamin B 12 is involved in the metabolism of methionine in sulfanilamide-inhibited E. coli .