The Experts below are selected from a list of 237 Experts worldwide ranked by ideXlab platform
Valeria C Culotta - One of the best experts on this subject based on the ideXlab platform.
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the yeast copper zinc superoxide dismutase and the pentose phosphate pathway play overlapping roles in oxidative stress protection
Journal of Biological Chemistry, 1996Co-Authors: Kimberly Hudak Slekar, Daniel J Kosman, Valeria C CulottaAbstract:Next Section Abstract In Saccharomyces cerevisiae, loss of cytosolic superoxide dismutase (Sod1) results in several air-dependent mutant phenotypes, including methionine Auxotrophy and oxygen sensitivity. Here we report that these two sod1Δ phenotypes were specifically suppressed by elevated expression of the TKL1 gene, encoding transketolase of the pentose phosphate pathway. The apparent connection between Sod1 and the pentose phosphate pathway prompted an investigation of mutants defective in glucose-6-phosphate dehydrogenase (Zwf1), which catalyzes the rate-limiting NADPH-producing step of this pathway. We confirmed that zwf1Δ mutants are methionine auxotrophs and report that they also are oxygen-sensitive. We determined that a functional ZWF1 gene product was required for TKL1 to suppress sod1Δ, leading us to propose that increased flux through the oxidative reactions of the pentose phosphate pathway can rescue sod1 methionine Auxotrophy. To better understand this methionine growth requirement, we examined the sulfur compound requirements of sod1Δ and zwf1Δ mutants, and noted that these mutants exhibit the same apparent defect in sulfur assimilation. Our studies suggest that this defect results from the impaired redox status of aerobically grown sod1 and zwf1 mutants, implicating Sod1 and the pentose phosphate pathway as being critical for maintenance of the cellular redox state.
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The yeast copper/zinc superoxide dismutase and the pentose phosphate pathway play overlapping roles in oxidative stress protection.
Journal of Biological Chemistry, 1996Co-Authors: Kimberly Hudak Slekar, Daniel J Kosman, Valeria C CulottaAbstract:Next Section Abstract In Saccharomyces cerevisiae, loss of cytosolic superoxide dismutase (Sod1) results in several air-dependent mutant phenotypes, including methionine Auxotrophy and oxygen sensitivity. Here we report that these two sod1Δ phenotypes were specifically suppressed by elevated expression of the TKL1 gene, encoding transketolase of the pentose phosphate pathway. The apparent connection between Sod1 and the pentose phosphate pathway prompted an investigation of mutants defective in glucose-6-phosphate dehydrogenase (Zwf1), which catalyzes the rate-limiting NADPH-producing step of this pathway. We confirmed that zwf1Δ mutants are methionine auxotrophs and report that they also are oxygen-sensitive. We determined that a functional ZWF1 gene product was required for TKL1 to suppress sod1Δ, leading us to propose that increased flux through the oxidative reactions of the pentose phosphate pathway can rescue sod1 methionine Auxotrophy. To better understand this methionine growth requirement, we examined the sulfur compound requirements of sod1Δ and zwf1Δ mutants, and noted that these mutants exhibit the same apparent defect in sulfur assimilation. Our studies suggest that this defect results from the impaired redox status of aerobically grown sod1 and zwf1 mutants, implicating Sod1 and the pentose phosphate pathway as being critical for maintenance of the cellular redox state.
Kimberly Hudak Slekar - One of the best experts on this subject based on the ideXlab platform.
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the yeast copper zinc superoxide dismutase and the pentose phosphate pathway play overlapping roles in oxidative stress protection
Journal of Biological Chemistry, 1996Co-Authors: Kimberly Hudak Slekar, Daniel J Kosman, Valeria C CulottaAbstract:Next Section Abstract In Saccharomyces cerevisiae, loss of cytosolic superoxide dismutase (Sod1) results in several air-dependent mutant phenotypes, including methionine Auxotrophy and oxygen sensitivity. Here we report that these two sod1Δ phenotypes were specifically suppressed by elevated expression of the TKL1 gene, encoding transketolase of the pentose phosphate pathway. The apparent connection between Sod1 and the pentose phosphate pathway prompted an investigation of mutants defective in glucose-6-phosphate dehydrogenase (Zwf1), which catalyzes the rate-limiting NADPH-producing step of this pathway. We confirmed that zwf1Δ mutants are methionine auxotrophs and report that they also are oxygen-sensitive. We determined that a functional ZWF1 gene product was required for TKL1 to suppress sod1Δ, leading us to propose that increased flux through the oxidative reactions of the pentose phosphate pathway can rescue sod1 methionine Auxotrophy. To better understand this methionine growth requirement, we examined the sulfur compound requirements of sod1Δ and zwf1Δ mutants, and noted that these mutants exhibit the same apparent defect in sulfur assimilation. Our studies suggest that this defect results from the impaired redox status of aerobically grown sod1 and zwf1 mutants, implicating Sod1 and the pentose phosphate pathway as being critical for maintenance of the cellular redox state.
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The yeast copper/zinc superoxide dismutase and the pentose phosphate pathway play overlapping roles in oxidative stress protection.
Journal of Biological Chemistry, 1996Co-Authors: Kimberly Hudak Slekar, Daniel J Kosman, Valeria C CulottaAbstract:Next Section Abstract In Saccharomyces cerevisiae, loss of cytosolic superoxide dismutase (Sod1) results in several air-dependent mutant phenotypes, including methionine Auxotrophy and oxygen sensitivity. Here we report that these two sod1Δ phenotypes were specifically suppressed by elevated expression of the TKL1 gene, encoding transketolase of the pentose phosphate pathway. The apparent connection between Sod1 and the pentose phosphate pathway prompted an investigation of mutants defective in glucose-6-phosphate dehydrogenase (Zwf1), which catalyzes the rate-limiting NADPH-producing step of this pathway. We confirmed that zwf1Δ mutants are methionine auxotrophs and report that they also are oxygen-sensitive. We determined that a functional ZWF1 gene product was required for TKL1 to suppress sod1Δ, leading us to propose that increased flux through the oxidative reactions of the pentose phosphate pathway can rescue sod1 methionine Auxotrophy. To better understand this methionine growth requirement, we examined the sulfur compound requirements of sod1Δ and zwf1Δ mutants, and noted that these mutants exhibit the same apparent defect in sulfur assimilation. Our studies suggest that this defect results from the impaired redox status of aerobically grown sod1 and zwf1 mutants, implicating Sod1 and the pentose phosphate pathway as being critical for maintenance of the cellular redox state.
William R. Jacobs - One of the best experts on this subject based on the ideXlab platform.
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rational design of biosafety level 2 approved multidrug resistant strains of mycobacterium tuberculosis through nutrient Auxotrophy
Mbio, 2018Co-Authors: Catherine Vilcheze, Torin R Weisbrod, Jacqueline Copeland, Tracy L Keiser, Jacqueline Washington, Paras Jain, Adel A Malek, Brian Weinrick, William R. JacobsAbstract:Multidrug-resistant (MDR) tuberculosis, defined as tuberculosis resistant to the two first-line drugs isoniazid and rifampin, poses a serious problem for global tuberculosis control strategies. Lack of a safe and convenient model organism hampers progress in combating the spread of MDR strains of Mycobacterium tuberculosis We reasoned that auxotrophic MDR mutants of M. tuberculosis would provide a safe means for studying MDR M. tuberculosis without the need for a biosafety level 3 (BSL3) laboratory. Two different sets of triple auxotrophic mutants of M. tuberculosis were generated, which were auxotrophic for the nutrients leucine, pantothenate, and arginine or for leucine, pantothenate, and methionine. These triple auxotrophic strains retained their acid-fastness, their ability to generate both a drug persistence phenotype and drug-resistant mutants, and their susceptibility to plaque-forming mycobacterial phages. MDR triple auxotrophic mutants were obtained in a two-step fashion, selecting first for solely isoniazid-resistant or rifampin-resistant mutants. Interestingly, selection for isoniazid-resistant mutants of the methionine auxotroph generated isolates with single point mutations in katG, which encodes an isoniazid-activating enzyme, whereas similar selection using the arginine auxotroph yielded isoniazid-resistant mutants with large deletions in the chromosomal region containing katG These M. tuberculosis MDR strains were readily sterilized by second-line tuberculosis drugs and failed to kill immunocompromised mice. These strains provide attractive candidates for M. tuberculosis biology studies and drug screening outside the BSL3 facility.IMPORTANCE Elimination of Mycobacterium tuberculosis, the bacterium causing tuberculosis, requires enhanced understanding of its biology in order to identify new drugs against drug-susceptible and drug-resistant M. tuberculosis as well as uncovering novel pathways that lead to M. tuberculosis death. To circumvent the need for a biosafety level 3 (BSL3) laboratory when conducting research on M. tuberculosis, we have generated drug-susceptible and drug-resistant triple auxotrophic strains of M. tuberculosis suitable for use in a BSL2 laboratory. These strains originate from a double auxotrophic M. tuberculosis strain, H37Rv ΔpanCD ΔleuCD, which was reclassified as a BSL2 strain based on its lack of lethality in immunocompromised and immunocompetent mice. A third Auxotrophy (methionine or arginine) was introduced via deletion of metA or argB, respectively, since M. tuberculosis ΔmetA and M. tuberculosis ΔargB are unable to survive amino acid Auxotrophy and infect their host. The resulting triple auxotrophic M. tuberculosis strains retained characteristics of M. tuberculosis relevant for most types of investigations.
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biosynthesis of diaminopimelate the precursor of lysine and a component of peptidoglycan is an essential function of mycobacterium smegmatis
Journal of Bacteriology, 1996Co-Authors: Martin S Pavelka, William R. JacobsAbstract:Diaminopimelate (DAP) is a unique metabolite used for both the biosynthesis of lysine in bacteria and the construction of the peptidoglycan of many species of bacteria, including mycobacteria. DAP is synthesized by bacteria as part of the aspartate amino acid family, which includes methionine, threonine, isoleucine, and lysine. Aspartokinase, the first enzyme in this pathway, is encoded by the ask gene in mycobacteria. Previous attempts to disrupt this gene in Mycobacterium smegmatis were unsuccessful, even when the cells were supplied with all the members of the aspartate family, suggesting that unlike other bacteria, mycobacteria may have an absolute requirement for this pathway even when growing in rich medium containing DAP. The purpose of this study was to determine if the ask gene and the aspartate pathway are essential to M. smegmatis. This study describes a test for gene essentiality in mycobacteria, utilizing a counterselectable marker (streptomycin resistance) in conjunction with a specially constructed merodiploid strain. We have used this system to show that the ask gene could not be disrupted in wild-type M. smegmatis, using selective rich medium supplemented with DAP unless there was an extra copy of ask provided elsewhere in the chromosome. Disruption of ask was also possible in a lysine auxotroph incapable of converting DAP to lysine. The ask mutant, mc21278 (ask1::aph), exhibits multiple Auxotrophy (Met-, Thr-, DAP-, and Lys-) and is complemented by the ask gene. This is the first description of DAP Auxotrophy in mycobacteria. The ask mutant lyses when deprived of DAP in culture, a characteristic which can be exploited for the reproducible preparation of protoplasts and mycobacterial extracts. The evidence presented here indicates that the aspartate pathway is essential to M. smegmatis and that DAP is the essential product of this pathway.
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Leucine Auxotrophy restricts growth of Mycobacterium bovis BCG in macrophages.
Infection and immunity, 1996Co-Authors: Franz Christoph Bange, Amanda C. Brown, William R. JacobsAbstract:The ability of slow-growing mycobacteria to replicate within host mononuclear phagocytes is thought to be central to the pathogenesis of mycobacterial infection. However, because of the lack of a mycobacterial mutant defective for intracellular replication, it has not been possible to test this hypothesis directly. Previously, we showed that a BCG leucine auxotroph with a transposon disruption of the leuD gene is unable to grow in mice. Here we demonstrate that this mutant is also incapable of replicating within cultured macrophages in vitro. Complementation of the leuD mutation with the leuCD genes of Escherichia coli restored wild-type levels of growth in macrophages, establishing that the defect for intracellular replication was due to leucine Auxotrophy per se and not to a polar effect of the transposon insertion on an adjacent gene. These results suggest that the inability of the leucine auxotroph to grow in mice was due to its sequestration, after phagocytosis, in an intracellular compartment from which it could not obtain leucine.
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in vivo growth characteristics of leucine and methionine auxotrophic mutants of mycobacterium bovis bcg generated by transposon mutagenesis
Infection and Immunity, 1995Co-Authors: R A Mcadam, Torin R Weisbrod, Jennifer L Martin, J D Scuderi, Amanda M Brown, Jeffrey D Cirillo, Barry R Bloom, William R. JacobsAbstract:Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen.
Alison G Smith - One of the best experts on this subject based on the ideXlab platform.
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algae acquire vitamin b12 through a symbiotic relationship with bacteria
Nature, 2005Co-Authors: Martin T Croft, A Lawrence, Evelyne Rauxdeery, Martin J Warren, Alison G SmithAbstract:Vitamin B12 (cobalamin) was identified nearly 80 years ago as the anti-pernicious anaemia factor in liver, and its importance in human health and disease has resulted in much work on its uptake, cellular transport and utilization. Plants do not contain cobalamin because they have no cobalamin-dependent enzymes. Deficiencies are therefore common in strict vegetarians, and in the elderly, who are susceptible to an autoimmune disorder that prevents its efficient uptake. In contrast, many algae are rich in vitamin B12, with some species, such as Porphyra yezoensis (Nori), containing as much cobalamin as liver. Despite this, the role of the cofactor in algal metabolism remains unknown, as does the source of the vitamin for these organisms. A survey of 326 algal species revealed that 171 species require exogenous vitamin B12 for growth, implying that more than half of the algal kingdom are cobalamin auxotrophs. Here we show that the role of vitamin B12 in algal metabolism is primarily as a cofactor for vitamin B12-dependent methionine synthase, and that cobalamin Auxotrophy has arisen numerous times throughout evolution, probably owing to the loss of the vitamin B12-independent form of the enzyme. The source of cobalamin seems to be bacteria, indicating an important and unsuspected symbiosis.
Donald A. Phillips - One of the best experts on this subject based on the ideXlab platform.
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biotin and other water soluble vitamins are key growth factors for alfalfa root colonization by rhizobium meliloti 1021
Molecular Plant-microbe Interactions, 1996Co-Authors: Wolfgang R Streit, Cecillia M. Joseph, Donald A. PhillipsAbstract:Rhizosphere growth limitations imposed on Rhizobium meliloti by availability of biotin, thiamine, and riboflavin were overcome by adding nanomolar amounts of these vitamins. Studies done with R. meliloti 1021 showed that both synthesis and uptake of biotin promote colonization of alfalfa roots. Two lines of evidence indicated that plant-derived biotin normally promotes root colonization: (i) adding avidin significantly (P less than or equal to 0.01) reduced rhizosphere growth of R. meliloti 1021, and (ii) growth of Tn5-induced biotin auxotrophs still increased 10-fold in the rhizosphere. Synthesis, however, is the more important source of biotin for R. meliloti 1021 because in root colonization tests biotin auxotrophs competed very poorly with the parent strain. Mutations conferring biotin Auxotrophy were closely linked on a single restriction fragment, and one was complemented with the Escherichia coli bio operon. Initial nucleotide sequencing and DNA-DNA hybridization tests showed the biotin synthesis genes in R. meliloti are quite different from those in E. coli.
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biotin and other water soluble vitamins are key growth factors for alfalfa root colonization by rhizobium meliloti 1021
Molecular Plant-microbe Interactions, 1996Co-Authors: Wolfgang R Streit, Cecillia M. Joseph, Donald A. PhillipsAbstract:Rhizosphere growth limitations imposed on Rhizobium meliloti by availability of biotin, thiamine, and riboflavin were overcome by adding nanomolar amounts of these vitamins. Studies done with R. meliloti 1021 showed that both synthesis and uptake of biotin promote colonization of alfalfa roots. Two lines of evidence indicated that plant-derived biotin normally promotes root colonization: (i) adding avidin significantly (P < or = 0.01) reduced rhizosphere growth of R meliloti 1021, and (ii) growth of Tn5-induced biotin auxotrophs still increased 10-fold in the rhizosphere. Synthesis, however, is the more important source of biotin for R. meliloti 1021 because in root colonization tests biotin auxotrophs competed very poorly with the parent strain. Mutations conferring biotin Auxotrophy were closely linked on a single restriction fragment, and one was complemented with the Escherichia coli bio operon. Initial nucleotide sequencing and DNA-DNA hybridization tests showed the biotin synthesis genes in R. meliloti are quite different from those in E. coli.