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Sample records for kadi ilves kadri

  1. Antipolarity in the ilv Operon of Escherichia coli K-12

    PubMed Central

    Wechsler, James A.; Adelberg, E. A.

    1969-01-01

    The genes governing three of the enzymes of the isoleucine-valine biosynthetic pathway form the operon: operator-ilvA-ilvD-ilvE. The enzymes are: ilvA, l-threonine deaminase; ilvD, dihydroxy acid dehydrase; and ilvE, transaminase B. A nonsense mutation in the ilvD gene (D-ochre) and a nonsense mutation in the ilvE gene (E-amber) affect the properties of the proximal gene product, l-threonine deaminase (TD), in addition to inactivating the enzymes produced by the genes in which the mutations have occurred. The D-ochre mutation causes TD to move in diffusion and gel filtration experiments as though it were 30% smaller than the wild-type enzyme. The E-amber mutation causes TD to move in similar experiments as though it were much larger than the wild-type enzyme. Both mutations completely abolish the sensitivity of TD to l-isoleucine, the normal feedback inhibitor of the wild-type enzyme. The effects of the nonsense mutations on TD can be reversed in three ways: by genetic reversion of the D-ochre mutation; by treatment of the altered enzymes with 3.0 m urea; and by forming a heterozygous diploid, containing the wild-type allele as well as the mutant allele of ilvD or ilvE. The results suggest that the subunits of TD undergo abnormal aggregation in the presence of the partial polypeptides produced by the mutant alleles of ilvD or ilvE; multi-enzyme aggregates in extracts of wild type, however, could not be detected. PMID:4892370

  2. Towards diacetyl-less brewers' yeast. Influence of ilv2 and ilv5 mutations.

    PubMed

    Gjermansen, C; Nilsson-Tillgren, T; Petersen, J G; Kielland-Brandt, M C; Sigsgaard, P; Holmberg, S

    1988-01-01

    During alcoholic fermentations, the off-flavour compound diacetyl is formed non-enzymatically from acetolactate leaking out from the cells. Acetolactate is an intermediate in the biosynthesis of valine. In beer fermentation, the amount of diacetyl is reduced to acceptable levels during maturation. A reduction of the time needed for maturation may be achieved by the use of a brewing yeast that produces less diacetyl. Saccharomyces cerevisiae laboratory strains with an inactive ilv2 gene can not form acetolactate, while ilv5 strains, blocked in the subsequent step, leak acetolactate in high amounts. Induction of recessive mutations in production strains of Saccharomyces carlsbergensis has not yet been achieved, as the yeast is polyploid and possibly a hybrid between S. cerevisiae and another Saccharomyces species. Thus, all chromosomes investigated so far are present in at least two genetically different versions. Genetic and molecular analysis has shown that the brewing yeast is structurally heterozygous for ILV2 and ILV5. Genetic modification of brewers' yeast to reduce diacetyl formation is being carried out by mutation of ILV2. Deletion mutations in both ILV2 alleles have been constructed in vitro to be used for gene replacement in the brewing strain. In addition, partial inactivation of the ILV2 function is carried out by selecting spontaneous dominant mutations resistant to the herbicide sulfometuron methyl. Among these mutants some produce only half the amount of diacetyl compared to the parental strain. An alternative way to reduce diacetyl production might be to increase the activity of the ILV5 gene product. Model experiments in S. cerevisiae show that the presence of the ILV5 gene on a 2-micron based multi-copy vector can reduce the diacetyl production by half.

  3. Acetohydroxyacid synthase FgIlv2 and FgIlv6 are involved in BCAA biosynthesis, mycelial and conidial morphogenesis, and full virulence in Fusarium graminearum

    PubMed Central

    Liu, Xin; Han, Qi; Xu, Jianhong; Wang, Jian; Shi, Jianrong

    2015-01-01

    In this study, we characterized FgIlv2 and FgIlv6, the catalytic and regulatory subunits of acetohydroxyacid synthase (AHAS) from the important wheat head scab fungus Fusarium graminearum. AHAS catalyzes the first common step in the parallel pathways toward branched-chain amino acids (BCAAs: isoleucine, leucine, valine) and is the inhibitory target of several commercialized herbicides. Both FgILV2 and FgILV6 deletion mutants were BCAA-auxotrophic and showed reduced aerial hyphal growth and red pigmentation when cultured on PDA plates. Conidial formation was completely blocked in the FgILV2 deletion mutant ΔFgIlv2-4 and significantly reduced in the FgILV6 deletion mutant ΔFgIlv6-12. The auxotrophs of ΔFgIlv2-4 and ΔFgIlv6-12 could be restored by exogenous addition of BCAAs but relied on the designated nitrogen source the medium contained. Deletion of FgILV2 or FgILV6 also leads to hypersensitivity to various cellular stresses and reduced deoxynivalenol production. ΔFgIlv2-4 lost virulence completely on flowering wheat heads, whereas ΔFgIlv6-12 could cause scab symptoms in the inoculated spikelet but lost its aggressiveness. Taken together, our study implies the potential value of antifungals targeting both FgIlv2 and FgIlv6 in F. graminearum. PMID:26552344

  4. Acetohydroxyacid synthase FgIlv2 and FgIlv6 are involved in BCAA biosynthesis, mycelial and conidial morphogenesis, and full virulence in Fusarium graminearum.

    PubMed

    Liu, Xin; Han, Qi; Xu, Jianhong; Wang, Jian; Shi, Jianrong

    2015-01-01

    In this study, we characterized FgIlv2 and FgIlv6, the catalytic and regulatory subunits of acetohydroxyacid synthase (AHAS) from the important wheat head scab fungus Fusarium graminearum. AHAS catalyzes the first common step in the parallel pathways toward branched-chain amino acids (BCAAs: isoleucine, leucine, valine) and is the inhibitory target of several commercialized herbicides. Both FgILV2 and FgILV6 deletion mutants were BCAA-auxotrophic and showed reduced aerial hyphal growth and red pigmentation when cultured on PDA plates. Conidial formation was completely blocked in the FgILV2 deletion mutant ΔFgIlv2-4 and significantly reduced in the FgILV6 deletion mutant ΔFgIlv6-12. The auxotrophs of ΔFgIlv2-4 and ΔFgIlv6-12 could be restored by exogenous addition of BCAAs but relied on the designated nitrogen source the medium contained. Deletion of FgILV2 or FgILV6 also leads to hypersensitivity to various cellular stresses and reduced deoxynivalenol production. ΔFgIlv2-4 lost virulence completely on flowering wheat heads, whereas ΔFgIlv6-12 could cause scab symptoms in the inoculated spikelet but lost its aggressiveness. Taken together, our study implies the potential value of antifungals targeting both FgIlv2 and FgIlv6 in F. graminearum. PMID:26552344

  5. Transcriptional activity of the transposable element Tn10 in the Salmonella typhimurium ilvGEDA operon.

    PubMed Central

    Blazey, D L; Burns, R O

    1982-01-01

    Polarity of Tn10 insertion mutations in the Salmonella typhimurium ilvGEDA operon depends on both the location and the orientation of the Tn10 element. One orientation of Tn10 insertions in ilvG and ilvE permits low-level expression of the downstream ilvEDA and ilvDA genes, respectively. Our analysis of Salmonella ilv recombinant plasmids shows that this residual ilv expression must result from Tn10-directed transcription and does not reflect the presence of internal promoters in the ilvGEDA operon, as was previously suggested. The opposite orientation of Tn10 insertion in ilvE prevents ilvDA expression, indicating that only one end of Tn10 is normally active in transcribing adjacent genes. Both orientations of Tn10 insertion in ilvD exert absolute polarity on ilvA expression. Expression of ilvA is known to be dependent on effective translation of ilvD, perhaps reflecting the lack of a ribosome binding site proximal to the ilvA sequence. Therefore, recognition of the ability of Tn10 to promote transcription of contiguous genes in the ilvGEDA operon apparently requires the presence of associated ribosome binding sites. PMID:6289328

  6. Organization and regulation of the ilvGEDA operon in Salmonella typhimurium LT2.

    PubMed Central

    Berg, C M; Shaw, K J

    1981-01-01

    A total of 102 isoleucine- and isoleucine-valine-requiring (ilv) mutants induced by insertion of the transposable element Tn10 have been classified to cistron by growth requirement, cross-feeding behavior, and enzyme assays. The mutations are in a polycistronic operon transcribed in the order ilvGEDA and in a monocistronic operon ilvC. Analysis of distal gene expression in these polar insertion mutants revealed the existence of two constitutive interval promoters, one preceding ilvE and the other preceding ilvD. Images PMID:7007356

  7. Physical organization of the ilvEDAC genes of Escherichia coli strain K-12

    PubMed Central

    McCorkle, George M.; Leathers, Timothy D.; Umbarger, H. E.

    1978-01-01

    We have determined the physical location of the ilvEDAC genes on the restriction cleavage map of the ilv region of Escherichia coli K-12 by two methods: (i) heteroduplex and endonuclease cleavage analysis of hybrid phages carrying genetically defined parts of the ilv cluster and (ii) complementation analysis and enzyme assays to determine ilv gene expression from hybrid plasmids containing DNA restriction fragments of the transducing phage λh80dilv. The ilvEDA and ilvC operons occupy 2.4 and 0.9 megadalton sequences of DNA, respectively, and are separated by a region of 0.6-0.75 megadalton. The ilvD region, specifying dihydroxy acid dehydrase, has a maximum coding capacity of about 55,000 daltons of polypeptide. Our results confirm that ilvC is transcribed clockwise on the E. coli K-12 map, in the same direction as ilaEDA. A secondary λ attachment site within ilvC has been located on a small (0.45 megadalton) EcoRI fragment. Our results are compared to other physical studies of ilv DNA. PMID:343113

  8. A New Map Location for the ilvB Locus of ESCHERICHIA COLI

    PubMed Central

    Newman, Thomas C.; Levinthal, Mark

    1980-01-01

    We isolated, in E. coli K12, new alleles of the ilvB locus, the structural gene for acetolactate synthase isoenzyme I, and showed them to map at or near the ilvB619 site. The map position of the ilvB locus was redetermined because plasmids containing the ilvC-cya portion of the chromosome did not complement mutations at the ilvB locus. Furthermore, diploids for the ilvEDAC genes formed with these plasmids in an ilvHI background facilitated the isolation of the new ilvB alleles. The ilvB locus was remapped and found to be located at 81.5 minutes, between the uhp and dnaA loci. This location was determined by two- and three-point transductional crosses, deletion mapping and complementation with newly isolated plasmids. One of the new alleles of the ilvB gene is a mu-1 insertion. When present in the donor strain, this allele interferes with the linkage of genes flanking the mu-1 insertion, as well as the linkage of genes to either side of the mu-1 insertion. PMID:7009323

  9. The 1.9 A Structure of the Branched-Chain Amino-Acid Transaminase (IlvE) from Mycobacterium tuberculosis

    SciTech Connect

    Tremblay, L.; Blanchard, J

    2009-01-01

    Unlike mammals, bacteria encode enzymes that synthesize branched-chain amino acids. The pyridoxal 5'-phosphate-dependent transaminase performs the final biosynthetic step in these pathways, converting keto acid precursors into {alpha}-amino acids. The branched-chain amino-acid transaminase from Mycobacterium tuberculosis (MtIlvE) has been crystallized and its structure has been solved at 1.9 {angstrom} resolution. The MtIlvE monomer is composed of two domains that interact to form the active site. The biologically active form of IlvE is a homodimer in which each monomer contributes a substrate-specificity loop to the partner molecule. Additional substrate selectivity may be imparted by a conserved N-terminal Phe30 residue, which has previously been observed to shield the active site in the type IV fold homodimer. The active site of MtIlvE contains density corresponding to bound PMP, which is likely to be a consequence of the presence of tryptone in the crystallization medium. Additionally, two cysteine residues are positioned at the dimer interface for disulfide-bond formation under oxidative conditions. It is unknown whether they are involved in any regulatory activities analogous to those of the human mitochondrial branched-chain amino-acid transaminase.

  10. FgIlv5 is required for branched-chain amino acid biosynthesis and full virulence in Fusarium graminearum.

    PubMed

    Liu, Xin; Wang, Jian; Xu, Jianhong; Shi, Jianrong

    2014-04-01

    In this study, we characterized FgIlv5, a homologue of the Saccharomyces cerevisiae keto-acid reductoisomerase (KARI) from the important wheat head scab fungus Fusarium graminearum. KARI is a key enzyme in the branched-chain amino acid (BCAA, including leucine, isoleucine and valine) biosynthetic pathway that exists in a variety of organisms from bacteria to fungi and higher plants, but not in mammals. The FgILV5 deletion mutant ΔFgIlv5-4 failed to grow when the culture medium was nutritionally limited for BCAAs. When grown on potato-dextrose agar plates, ΔFgIlv5-4 exhibited a significant decrease in aerial hyphae formation and red pigmentation. Conidia formation was also blocked in ΔFgIlv5-4. Exogenous addition of 1 mM isoleucine and valine was able to rescue the defects of mycelial growth and conidial morphogenesis. Cellular stress assays showed that ΔFgIlv5-4 was more sensitive to osmotic and oxidative stresses than the wild-type strain. In addition, virulence of ΔFgIlv5-4 was dramatically reduced on wheat heads, and a low level of deoxynivalenol production was detected in ΔFgIlv5-4 in wheat kernels. The results of this study indicate that FgIlv5 is involved in valine and isoleucine biosynthesis and is required for full virulence in F. graminearum.

  11. Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin.

    PubMed

    Kingsbury, Joanne M; McCusker, John H

    2010-03-01

    The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Delta mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Delta mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Delta mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Delta starvation-cidal defects in either species, the cidal phenotype was not due to alpha-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Delta starvation viability, it increased Ca. albicans ilv1Delta and ilv2Delta viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Delta mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Delta and ilv2 Delta viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal

  12. Analysis and comparison of the internal promoter, pE, of the ilvGMEDA operons from Escherichia coli K-12 and Salmonella typhimurium.

    PubMed Central

    Lopes, J M; Lawther, R P

    1986-01-01

    It was previously determined that the distal portion of the ilvGMEDA operon was expressed despite the insertion of transposons into ilvG and ilvE. This observation suggested the existence of internal promoters upstream of ilvE (pE) and ilvD (pD). The internal promoter pE, responsible for part of ilvEDA expression, has been analyzed both in vivo and in vitro. Our results indicate that: pE exists in both E. coli K-12 and S. typhimurium; pE is located in the distal end of the ilvM coding sequence; the pE sequence is highly conserved in the two bacteria; the amino acid sequence of the ilvM gene product is 93% homologous between the two bacteria; transcription from pE can be demonstrated both in vivo and in vitro; the efficiency of pE is essentially equivalent in the two bacteria. Images PMID:3008097

  13. The product of the Rhizobium meliloti ilvC gene is required for isoleucine and valine synthesis and nodulation of alfalfa.

    PubMed Central

    Aguilar, O M; Grasso, D H

    1991-01-01

    Tn5-induced mutants of Rhizobium meliloti that require the amino acids isoleucine and valine for growth on minimal medium were studied. In one mutant, 1028, the defect is associated with an inability to induce nodules on alfalfa. The Tn5 mutation in 1028 is located in a chromosomal 5.5-kb EcoRI fragment. Complementation analysis with cloned DNA indicated that 2.0 kb of DNA from the 5.5-kb EcoRI fragment restored the wild-type phenotype in the Ilv- Nod- mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to those for Escherichia coli IlvC and Saccharomyces cerevisiae Ilv5. Genes ilvC and ilv5 code for the enzyme acetohydroxy acid isomeroreductase (isomeroreductase), the second enzyme in the parallel pathways for the biosynthesis of isoleucine and valine. Enzymatic assays confirmed that strain 1028 was a mutant defective in isomeroreductase activity. In addition, it was shown that the ilvC genes of Rhizobium meliloti and E. coli are functionally equivalent. We demonstrated that in ilvC mutant 1028 the common nodulation genes nodABC are not activated by the inducer luteolin. E. coli ilvC complemented both defective properties (Ilv- and Nod-) found in mutant 1028. These findings demonstrate that R. meliloti requires an active isomeroreductase enzyme for successful nodulation of alfalfa. PMID:1744032

  14. Overexpression of the truncated version of ILV2 enhances glycerol production in Saccharomyces cerevisiae.

    PubMed

    Murashchenko, Lidiia; Abbas, Charles; Dmytruk, Kostyantyn; Sibirny, Andriy

    2016-08-01

    Acetolactate synthase is a mitochondrial enzyme that catalyses the conversion of two pyruvate molecules to an acetolactate molecule with release of carbon dioxide. The overexpression of the truncated version of the corresponding gene, ILV2, that codes for presumably cytosolic acetolactate synthase in the yeast Saccharomyces cerevisiae, led to a decrease in intracellular pyruvate concentration. This recombinant strain was also characterized by a four-fold increase in glycerol production, with a concomitant 1.8-fold reduction in ethanol production, when compared to that of the wild-type strain under anaerobic conditions in a glucose alcoholic fermentation. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26990811

  15. Construction of self-cloning bottom-fermenting yeast with low vicinal diketone production by the homo-integration of ILV5.

    PubMed

    Kusunoki, K; Ogata, T

    2012-10-01

    The vicinal diketones (VDK), such as diacetyl and 2,3-pentandione, impart an unpleasant butter-like flavour to beer. Typically, these are required to be reduced below the flavour thresholds during the maturation (lagering) stages of the brewing process. To shorten beer maturation time, we constructed a self-cloning, bottom-fermenting yeast with low VDK production by integrating ILV5, a gene encoding a protein that metabolizes α-acetolactate and α-aceto-α-hydroxybutyrate (precursors of VDK). A DNA fragment containing Saccharomyces cerevisiae-type ILV5 was inserted upstream of S. cerevisiae-type ILV2 in bottom-fermenting yeast to construct self-cloning strains with an increased copy number of ILV5. Via transformation, ILV2 was replaced with the sulfometuron methyl (SM) resistance gene SMR1B, which differs by a single nucleotide, to create SM-resistant transformants. The wort fermentation test, using the SC-ILV5-homo inserted transformant, confirmed a consecutive reduction in VDK and a shortening period during which VDK was reduced to within the threshold. The concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, 1-propanol, isobutyl alcohol and active isoamyl alcohol (flavour components) were not changed when compared with the parent strain. We successfully constructed self-cloning brewer's yeast in which SC-ILV5 was homo-inserted. Using the transformed yeast, the concentration of VDK in fermenting wort was reduced, whereas the concentrations of flavour components were not affected. This genetically stable, low VDK-producing, self-cloning bottom-fermenting yeast would contribute to the shortening of beer maturation time without affecting important flavour components produced by brewer's yeast. PMID:23038161

  16. Identification of Sc-type ILV6 as a target to reduce diacetyl formation in lager brewers' yeast.

    PubMed

    Duong, C T; Strack, L; Futschik, M; Katou, Y; Nakao, Y; Fujimura, T; Shirahige, K; Kodama, Y; Nevoigt, E

    2011-11-01

    Diacetyl causes an unwanted buttery off-flavor in lager beer. It is spontaneously generated from α-acetolactate, an intermediate of yeast's valine biosynthesis released during the main beer fermentation. Green lager beer has to undergo a maturation process lasting two to three weeks in order to reduce the diacetyl level below its taste-threshold. Therefore, a reduction of yeast's α-acetolactate/diacetyl formation without negatively affecting other brewing relevant traits has been a long-term demand of brewing industry. Previous attempts to reduce diacetyl production by either traditional approaches or rational genetic engineering had different shortcomings. Here, three lager yeast strains with marked differences in diacetyl production were studied with regard to gene copy numbers as well as mRNA abundances under conditions relevant to industrial brewing. Evaluation of data for the genes directly involved in the valine biosynthetic pathway revealed a low expression level of Sc-ILV6 as a potential molecular determinant for low diacetyl formation. This hypothesis was verified by disrupting the two copies of Sc-ILV6 in a commercially used lager brewers' yeast strain, which resulted in 65% reduction of diacetyl concentration in green beer. The Sc-ILV6 deletions did not have any perceptible impact on beer taste. To our knowledge, this has been the first study exploiting natural diversity of lager brewers' yeast strains for strain optimization.

  17. Improvement of L-valine production at high temperature in Brevibacterium flavum by overexpressing ilvEBNrC genes.

    PubMed

    Hou, Xiaohu; Ge, Xiangyang; Wu, Di; Qian, He; Zhang, Weiguo

    2012-01-01

    Brevibacterium flavum ATCC14067 was engineered for L: -valine production by overexpression of different ilv genes; the ilvEBN(r)C genes from B. flavum NV128 provided the best candidate for L: -valine production. In traditional fermentation, L: -valine production reached 30.08 ± 0.92 g/L at 31°C in 72 h with a low conversion efficiency of 0.129 g/g. To further improve the L: -valine production and conversion efficiency based on the optimum temperatures of L: -valine biosynthesis enzymes (above 35°C) and the thermotolerance of B. flavum, the fermentation temperature was increased to 34, 37, and 40°C. As a result, higher metabolic rate and L: -valine biosynthesis enzymes activity were obtained at high temperature, and the maximum L: -valine production, conversion efficiency, and specific L: -valine production rate reached 38.08 ± 1.32 g/L, 0.241 g/g, and 0.133 g g(-1) h(-1), respectively, at 37°C in 48 h fermentation. The strategy for enhancing L: -valine production by overexpression of key enzymes in thermotolerant strains may provide an alternative approach to enhance branched-chain amino acids production with other strains.

  18. Factors enhancing L-valine production by the growth-limited L-isoleucine auxotrophic strain Corynebacterium glutamicum DeltailvA DeltapanB ilvNM13 (pECKAilvBNC).

    PubMed

    Denina, Ilze; Paegle, Longina; Prouza, Marek; Holátko, Jiri; Pátek, Miroslav; Nesvera, Jan; Ruklisha, Maija

    2010-07-01

    Cell growth limitation is known to be an important condition that enhances L: -valine synthesis in Corynebacterium glutamicum recombinant strains with L: -isoleucine auxotrophy. To identify whether it is the limited availability of L: -isoleucine itself or the L: -isoleucine limitation-induced rel-dependent ppGpp-mediated stringent response that is essential for the enhancement of L: -valine synthesis in growth-limited C. glutamicum cells, we deleted the rel gene, thereby constructing a relaxed (rel (-) ) C. glutamicum DeltailvA DeltapanB Deltarel ilvNM13 (pECKAilvBNC) strain. Variations in enzyme activity and L: -valine synthesis in rel (+) and rel (-) strains under conditions of L: -isoleucine excess and limitation were investigated. A sharp increase in acetohydroxy acid synthase (AHAS) activity, a slight increase in acetohydroxyacid isomeroreductase (AHAIR) activity, and a dramatic increase in L: -valine synthesis were observed in both rel (+) and rel (-) cells exposed to L: -isoleucine limitation. Although the positive effect of induction of the stringent response on AHAS and AHAIR upregulation in cells was not confirmed, we found the stringent response to be beneficial for maintaining increased AHAS, dihydroxyacid dehydratase, and transaminase B activity and L: -valine synthesis in cells during the stationary growth phase.

  19. Reduced Transaminase B (IlvE) Activity Caused by the Lack of yjgF Is Dependent on the Status of Threonine Deaminase (IlvA) in Salmonella enterica Serovar Typhimurium

    PubMed Central

    Schmitz, George; Downs, Diana M.

    2004-01-01

    The YjgF/YER057c/UK114 family is a highly conserved class of proteins that is represented in the three domains of life. Thus far, a biochemical function demonstrated for these proteins in vivo or in vitro has yet to be defined. In several organisms, strains lacking a YjgF homolog have a defect in branched-chain amino acid biosynthesis. This study probes the connection between yjgF and isoleucine biosynthesis in Salmonella enterica. In strains lacking yjgF the specific activity of transaminase B, catalyzing the last step in the synthesis of isoleucine, was reduced. In the absence of yjgF, transaminase B activity could be restored by inhibiting threonine deaminase, the first enzymatic step in isoleucine biosynthesis. Strains lacking yjgF showed an increased sensitivity to sulfometruron methyl, a potent inhibitor of acetolactate synthase. Based on work described here and structural reports in the literature, we suggest a working model in which YjgF has a role in protecting the cell from toxic effects of imbalanced ketoacid pools. PMID:14729707

  20. Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism

    PubMed Central

    2011-01-01

    Background Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric n-butanol. Saccharomyces cerevisiae was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials. Results The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in anaerobic fermentation of glucose in mineral medium in S. cerevisiae. Isobutanol yield was further improved by twofold by the additional overexpression of BAT2, encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of ILV6, encoding the regulatory subunit of Ilv2, in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the ILV2 ILV3 ILV5 overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD) complex medium under aerobic conditions, respectively. Conclusions Overexpression of genes ILV2, ILV3, ILV5, and BAT2 in valine metabolism led to an increase in isobutanol production in S. cerevisiae. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of ILV2, ILV3, and ILV5 on isobutanol production. Aerobic cultivations of the ILV2 ILV3 ILV5 overexpression strain and the reference strain showed that supplying amino acids in cultivation media gave a substantial

  1. Mutation affecting regulation of synthesis of acetohydroxy acid synthetase in Escherichia coli K-12.

    PubMed Central

    Jackson, J H; Henderson, E K

    1975-01-01

    Altered regulation of synthesis of acetohydroxy acid synthetase (AHAS) was previously reported in a mutant of Escherichia coli strain K-12. The mutant strain, growing in minimal medium, exhibits a partial growth limiatation and derepression of AHAS, owing to deficient synthesis of isoleucine. The genetic lesion (ilvE503) causing the isoleucine limitation was shown to cause derepression of a valine-sensitive AHAS activity. The derepression effect of the ilvE503 mutation upon synthesis of AHAS was conclusively demonstrated by introducing both the ilvE503 allele and an altered AHAS (ilv-521) into the same cell. Evidence is presented that suggests the presence of multiple genetic regions for synthesis and control of the valine-sensitive AHAS activity. PMID:1089632

  2. Metabolic engineering of Corynebacterium glutamicum ATCC13869 for L-valine production.

    PubMed

    Chen, Cheng; Li, Yanyan; Hu, Jinyu; Dong, Xunyan; Wang, Xiaoyuan

    2015-05-01

    In this study, an L-valine-producing strain was developed from Corynebacterium glutamicum ATCC13869 through deletion of the three genes aceE, alaT and ilvA combined with the overexpression of six genes ilvB, ilvN, ilvC, lrp1, brnF and brnE. Overexpression of lrp1 alone increased L-valine production by 16-fold. Deletion of the aceE, alaT and ilvA increased L-valine production by 44-fold. Overexpression of the six genes ilvB, ilvN, ilvC, lrp1, brnE and brnF in the triple deletion mutant WCC003 further increased L-valine production. The strain WCC003/pJYW-4-ilvBNC1-lrp1-brnFE produced 243mM L-valine in flask cultivation and 437mM (51g/L) L-valine in fed-batch fermentation and lacked detectable amino-acid byproduct such as l-alanine and l-isoleucine that are usually found in the fermentation of L-valine-producing C. glutamicum.

  3. Oahu Groundwater Flow Model

    DOE Data Explorer

    Nicole Lautze

    2015-01-01

    Groundwater flow model for the island of Oahu. Data is from the following sources: Rotzoll, K., A.I. El-Kadi. 2007. Numerical Ground-Water Flow Simulation for Red Hill Fuel Storage Facilities, NAVFAC Pacific, Oahu, Hawaii - Prepared TEC, Inc. Water Resources Research Center, University of Hawaii, Honolulu.; Whittier, R.B., K. Rotzoll, S. Dhal, A.I. El-Kadi, C. Ray, G. Chen, and D. Chang. 2004. Hawaii Source Water Assessment Program Report – Volume VII – Island of Oahu Source Water Assessment Program Report. Prepared for the Hawaii Department of Health, Safe Drinking Water Branch. University of Hawaii, Water Resources Research Center. Updated 2008.; and Whittier, R. and A.I. El-Kadi. 2009. Human and Environmental Risk Ranking of Onsite Sewage Disposal Systems – Final. Prepared by the University of Hawaii, Dept. of Geology and Geophysics for the State of Hawaii Dept. of Health, Safe Drinking Water Branch. December 2009.

  4. Suppressors of a genetic regulatory mutation affecting isoleucine-valine biosynthesis in Escherichia coli K-12.

    PubMed Central

    Hahn, J E; Calhoun, D H

    1978-01-01

    Escherichia coli K-12 mutant PS187 carries a mutation, ilvA538, in the structural gene for the biosynthetic L-threonine deaminase that leads to a leucine-sensitive growth phenotype, an isoleucine- and leucine-hypersensitive L-threonine deaminase, and pleiotropic effects resulting in abnormally low and invariant expression of some of the isoleucine-valine biosynthetic enzymes. Fifty-eight derivatives of strain PS187 were isolated as resistant to growth inhibition by leucine, by valine, or by valine plus glycly-valine and were biochemically, genetically, and physiologically characterized. All of these derivatives produced the feedback-hypersensitive L-threonine deaminase, and thus presumably possess the ilvA538 allele of the parent strain. Elevated synthesis of L-threonine deaminase was observed in 41 of the 58 isolates. Among 18 strains analyzed genetically, only those with mutations linked to the ilv gene clusters at 83 min produced elevated levels of L-threonine deaminase. One of the strains, MSR91, isolated as resistant to valine plus glycyl-valine, was chosen for more detailed study. The locus in strain MSR91 conferring resistance was located in four factor crosses between ilvE and rbs, and is in or near the ilvO gene postulated to be a site controlling the expression of the ilvEDA genes. Synthesis of the ilvEDA gene products in strain MSR91 is constitutive and derepressed approximately 200-fold relative to the parent strain, indicating that the genetic regulatory effects of the ilvA538 allele have been suppressed. Strain MSR91 should be suitable for use in purification of the ilvA538 gene product, since enzyme synthesis is fully derepressed and the suppressor mutation is clearly not located within the ilvA gene. PMID:361682

  5. Three-factor reciprocal cross mapping of a gene that causes expression of feedback-resistant acetohydroxy acid synthase in Escherichia coli K-12.

    PubMed

    Jackson, J H; Davis, E J; Madu, A C; Braxter, S E

    1981-01-01

    The ilv-662 allele was previously identified as a mutation that caused acetohydroxy acid synthase activity to be resistant to feedback inhibition by valine (Davis et al. 1977). This allele was mapped between thr and leu by cotransduction analysis and labeled ilvJ. This report describes the mapping of ilvJ relative to genes that lie between thr and leu (ara, carA and pdxA) by three factor reciprocal cross analyses. We find that the probable gene order is thr-carA-pdxA-ilvJ-ara-leu. Although the phenotypic properties of ilvJ662 appear to be quite distinct from brnS, a gene reported to involve branched chain amino acid transport (Guardiola et al. 1974), we do not rule out possible allelism because of the uncertainty of the map position of brnS.

  6. Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes.

    PubMed

    Vogt, Michael; Haas, Sabine; Polen, Tino; van Ooyen, Jan; Bott, Michael

    2015-03-01

    2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l(-1)) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l(-1)  h(-1). Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol. PMID:25488800

  7. RidA Proteins Prevent Metabolic Damage Inflicted by PLP-Dependent Dehydratases in All Domains of Life

    PubMed Central

    Lambrecht, Jennifer A.; Schmitz, George E.; Downs, Diana M.

    2013-01-01

    ABSTRACT Pyridoxal 5′-phosphate (PLP) is a coenzyme synthesized by all forms of life. Relevant to the work reported here is the mechanism of the PLP-dependent threonine/serine dehydratases, which generate reactive enamine/imine intermediates that are converted to keto acids by members of the RidA family of enzymes. The RidA protein of Salmonella enterica serovar Typhimurium LT2 is the founding member of this broadly conserved family of proteins (formerly known as YjgF/YER057c/UK114). RidA proteins were recently shown to be enamine deaminases. Here we demonstrate the damaging potential of enamines in the absence of RidA proteins. Notably, S. enterica strains lacking RidA have decreased activity of the PLP-dependent transaminase B enzyme IlvE, an enzyme involved in branched-chain amino acid biosynthesis. We reconstituted the threonine/serine dehydratase (IlvA)-dependent inhibition of IlvE in vitro, show that the in vitro system reflects the mechanism of RidA function in vivo, and show that IlvE inhibition is prevented by RidA proteins from all domains of life. We conclude that 2-aminoacrylate (2AA) inhibition represents a new type of metabolic damage, and this finding provides an important physiological context for the role of the ubiquitous RidA family of enamine deaminases in preventing damage by 2AA. PMID:23386433

  8. MRA_1571 is required for isoleucine biosynthesis and improves Mycobacterium tuberculosis H37Ra survival under stress

    PubMed Central

    Sharma, Rishabh; Keshari, Deepa; Singh, Kumar Sachin; Yadav, Shailendra; Singh, Sudheer Kumar

    2016-01-01

    Threonine dehydratase is a pyridoxal 5-phosphate dependent enzyme required for isoleucine biosynthesis. Threonine dehydratase (IlvA) participates in conversion of threonine to 2-oxobutanoate and ammonia is released as a by-product. MRA_1571 is annotated to be coding for IlvA in Mycobacterium tuberculosis H37Ra (Mtb-Ra). We developed a recombinant (KD) Mtb-Ra strain by down-regulating IlvA. The growth studies on different carbon sources suggested reduced growth of KD compared to wild-type (WT), also, isoleucine concentration dependent KD growth restoration was observed. The expression profiling of IlvA suggested increased expression of IlvA during oxygen, acid and oxidative stress. In addition, KD showed reduced survival under pH, starvation, nitric oxide and peroxide stresses. KD was more susceptible to antimycobacterial agents such as streptomycin (STR), rifampicin (RIF) and levofloxacin (LVF), while, no such effect was noticeable when exposed to isoniazid. Also, an increase in expression of IlvA was observed when exposed to STR, RIF and LVF. The dye accumulation studies suggested increased permeability of KD to ethidium bromide and Nile Red as compared to WT. TLC and Mass studies confirmed altered lipid profile of KD. In summary down-regulation of IlvA affects Mtb growth, increases its susceptibility to stress and leads to altered cell wall lipid profile. PMID:27353854

  9. MRA_1571 is required for isoleucine biosynthesis and improves Mycobacterium tuberculosis H37Ra survival under stress.

    PubMed

    Sharma, Rishabh; Keshari, Deepa; Singh, Kumar Sachin; Yadav, Shailendra; Singh, Sudheer Kumar

    2016-01-01

    Threonine dehydratase is a pyridoxal 5-phosphate dependent enzyme required for isoleucine biosynthesis. Threonine dehydratase (IlvA) participates in conversion of threonine to 2-oxobutanoate and ammonia is released as a by-product. MRA_1571 is annotated to be coding for IlvA in Mycobacterium tuberculosis H37Ra (Mtb-Ra). We developed a recombinant (KD) Mtb-Ra strain by down-regulating IlvA. The growth studies on different carbon sources suggested reduced growth of KD compared to wild-type (WT), also, isoleucine concentration dependent KD growth restoration was observed. The expression profiling of IlvA suggested increased expression of IlvA during oxygen, acid and oxidative stress. In addition, KD showed reduced survival under pH, starvation, nitric oxide and peroxide stresses. KD was more susceptible to antimycobacterial agents such as streptomycin (STR), rifampicin (RIF) and levofloxacin (LVF), while, no such effect was noticeable when exposed to isoniazid. Also, an increase in expression of IlvA was observed when exposed to STR, RIF and LVF. The dye accumulation studies suggested increased permeability of KD to ethidium bromide and Nile Red as compared to WT. TLC and Mass studies confirmed altered lipid profile of KD. In summary down-regulation of IlvA affects Mtb growth, increases its susceptibility to stress and leads to altered cell wall lipid profile.

  10. Isla Vista virus: a genetically novel hantavirus of the California vole Microtus californicus.

    PubMed

    Song, W; Torrez-Martinez, N; Irwin, W; Harrison, F J; Davis, R; Ascher, M; Jay, M; Hjelle, B

    1995-12-01

    Prospect Hill virus (PH) was isolated from a meadow vole (Microtus pennsylvanicus) in 1982, and much of its genome has been sequenced. Hantaviruses of other New World microtine rodents have not been genetically characterized. We show that another Microtus species (the California vole M. californicus) from the United States is host to a genetically distinct PH-like hantavirus, Isla Vista virus (ILV). The nucleocapsid protein of ILV differs from that of PH by 11.1% and a portion of the G2 glycoprotein differs from that of PH by 19.6%. ILV antibodies were identified in five of 33 specimens of M. californicus collected in 1975 and 1994-1995. Enzymatic amplification studies showed that 1975 and 1994-1995 ILV genomes were highly similar. Secondary infection of Peromyscus californicus was identified in Santa Barbara County, California. A long-standing enzootic of a genetically distinct hantavirus lineage is present in California voles. PMID:8847529

  11. Regulation of Transaminase C Synthesis in Escherichia coli: Conditional Leucine Auxotrophy

    PubMed Central

    McGilvray, Derek; Umbarger, H. E.

    1974-01-01

    The regulation of synthesis of the valine-alanine-α-aminobutyrate transaminase (transaminase C) was studied in Escherichia coli mutants lacking the branched-chain amino acid transaminase (transaminase B). An investigation was made of two strains, CU2 and CU2002, each carrying the same transaminase B lesion but exhibiting different growth responses on a medium supplemented with branched-chain amino acids. Both had the absolute isoleucine requirement characteristic of ilvE auxotrophs, but growth of strain CU2 was stimulated by valine, whereas that of strain CU2002 was markedly inhibited by valine. Strain CU2002 behaved like a conditional leucine auxotroph in that the inhibition by valine was reversed by leucine. Results of enzymatic studies showed that synthesis of transaminase C was repressed by valine in strain CU2002 but not in strain CU2. Inhibition by valine in strain CU2002 appears to be the combined effect of repression on transaminase C synthesis and valine-dependent feedback inhibition of α-acetohydroxy acid synthase activity, causing α-ketoisovalerate (and hence leucine) limitation. The ilvE markers of strains CU2 and CU2002 were each transferred by transduction to a wild-type genetical background. All ilvE recombinants from both crosses resembled strain CU2002 and were inhibited by valine in the presence of isoleucine. Thus, strain CU2 carries an additional lesion that allows it to grow on a medium containing isoleucine plus valine. It is concluded that conditional leucine auxotrophy is characteristic of mutants carrying an ilvE lesion alone. PMID:4616947

  12. Hawaii Island Groundwater Flow Model

    DOE Data Explorer

    Nicole Lautze

    2015-01-01

    Groundwater flow model for Hawaii Island. Data is from the following sources: Whittier, R.B., K. Rotzoll, S. Dhal, A.I. El-Kadi, C. Ray, G. Chen, and D. Chang. 2004. Hawaii Source Water Assessment Program Report – Volume II – Island of Hawaii Source Water Assessment Program Report. Prepared for the Hawaii Department of Health, Safe Drinking Water Branch. University of Hawaii, Water Resources Research Center. Updated 2008; and Whittier, R. and A.I. El-Kadi. 2014. Human and Environmental Risk Ranking of Onsite Sewage Disposal Systems For the Hawaiian Islands of Kauai, Molokai, Maui, and Hawaii – Final. Prepared by the University of Hawaii, Dept. of Geology and Geophysics for the State of Hawaii Dept. of Health, Safe Drinking Water Branch. September 2014.

  13. East Maui Groundwater Flow Model

    SciTech Connect

    Nicole Lautze

    2015-01-01

    Groundwater flow model for East Maui. Data is from the following sources: Whittier, R. and A.I. El-Kadi. 2014. Human and Environmental Risk Ranking of Onsite Sewage Disposal Systems For the Hawaiian Islands of Kauai, Molokai, Maui, and Hawaii – Final. Prepared by the University of Hawaii, Dept. of Geology and Geophysics for the State of Hawaii Dept. of Health, Safe Drinking Water Branch. September 2014; and Whittier, R.B., K. Rotzoll, S. Dhal, A.I. El-Kadi, C. Ray, G. Chen, and D. Chang. 2004. Hawaii Source Water Assessment Program Report – Volume V – Island of Maui Source Water Assessment Program Report. Prepared for the Hawaii Department of Health, Safe Drinking Water Branch. University of Hawaii, Water Resources Research Center. Updated 2008.

  14. West Maui Groundwater Flow Model

    SciTech Connect

    Nicole Lautze

    2015-01-01

    Groundwater flow model for West Maui. Data is from the following sources: Whittier, R. and A.I. El-Kadi. 2014. Human and Environmental Risk Ranking of Onsite Sewage Disposal Systems For the Hawaiian Islands of Kauai, Molokai, Maui, and Hawaii – Final. Prepared by the University of Hawaii, Dept. of Geology and Geophysics for the State of Hawaii Dept. of Health, Safe Drinking Water Branch. September 2014; and Whittier, R.B., K. Rotzoll, S. Dhal, A.I. El-Kadi, C. Ray, G. Chen, and D. Chang. 2004. Hawaii Source Water Assessment Program Report – Volume V – Island of Maui Source Water Assessment Program Report. Prepared for the Hawaii Department of Health, Safe Drinking Water Branch. University of Hawaii, Water Resources Research Center. Updated 2008.

  15. Kauai Groundwater Flow Model

    DOE Data Explorer

    Nicole Lautze

    2015-01-01

    Groundwater flow model for Kauai. Data is from the following sources: Whittier, R. and A.I. El-Kadi. 2014. Human and Environmental Risk Ranking of Onsite Sewage Disposal Systems For the Hawaiian Islands of Kauai, Molokai, Maui, and Hawaii – Final. Prepared by the University of Hawaii, Dept. of Geology and Geophysics for the State of Hawaii Dept. of Health, Safe Drinking Water Branch. September 2014.; and Whittier, R.B., K. Rotzoll, S. Dhal, A.I. El-Kadi, C. Ray, G. Chen, and D. Chang. 2004. Hawaii Source Water Assessment Program Report – Volume IV – Island of Kauai Source Water Assessment Program Report. Prepared for the Hawaii Department of Health, Safe Drinking Water Branch. University of Hawaii, Water Resources Research Center. Updated 2015.

  16. Special Analysis: Atmospheric Dose Resulting from the Release of C14 from Reactor Moderator Deionizers in a Disposal Environment

    SciTech Connect

    Hiergesell, Robert A.; Swingle, Robert F.

    2005-08-18

    The proposed action of disposing of 52 moderator deionizer vessels within the ILV was evaluated in this SA. In particular, a detailed analysis of the release of {sup 14}C via the atmospheric pathway was conducted for these vessels since the major concern has been the nearly 20 Ci of {sup 14}C that is associated with each vessel. The more rigorous evaluation of the atmospheric pathway for {sup 14}C included incorporation of new information about the chemical availability of {sup 14}C when disposed in a grout/cement encapsulation environment, as will be the case in the ILV. This information was utilized to establish the source term for a 1-D numerical model to simulate the diffusion of {sup 14}CO{sub 2} from the ILV Waste Zone to the land surface. The results indicate a peak surface emanation rate from the entire ILV of 1.42E-08 Ci/yr with an associated dose of only 3.83E-05 mrem/yr to the Maximally Exposed Individual (MEI) at 100m. The fact that the atmospheric pathway exposure for {sup 14}C is controlled by chemical solubility limits for {sup 14}C between the solid waste, pore water and pore vapor within the disposal environment rather than the absolute inventory suggests that the establishment of specific facility limits is inappropriate. With the relaxation of the atmospheric pathway restriction, the groundwater pathway becomes the more restrictive in terms of disposing {sup 14}C or {sup 14}C{sub KB} within the ILV. Since the resin-based {sup 14}C of the 52 moderator deionizer vessels is highly similar to the {sup 14}C{sub KB} waste form, the inventory from the 52 deionizer vessels is compared against the groundwater limits for that waste form. The small groundwater pathway fraction (1.14E-05) calculated for the proposed inventory of the 52 moderator deionizer vessels indicates that the proposed action will have an insignificant impact with respect to possible exposures via the groundwater pathway. This investigation recommends that there be no ILV Atmospheric

  17. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

    PubMed Central

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-01-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  18. Escherichia coli W as a new platform strain for the enhanced production of L-valine by systems metabolic engineering.

    PubMed

    Park, Jin Hwan; Jang, Yu-Sin; Lee, Jeong Wook; Lee, Sang Yup

    2011-05-01

    A less frequently employed Escherichia coli strain W, yet possessing useful metabolic characteristics such as less acetic acid production and high L-valine tolerance, was metabolically engineered for the production of L-valine. The ilvA gene was deleted to make more pyruvate, a key precursor for L-valine, available for enhanced L-valine biosynthesis. The lacI gene was deleted to allow constitutive expression of genes under the tac or trc promoter. The ilvBN(mut) genes encoding feedback-resistant acetohydroxy acid synthase (AHAS) I and the L-valine biosynthetic ilvCED genes encoding acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and branched chain amino acid aminotransferase, respectively, were amplified by plasmid-based overexpression. The global regulator Lrp and L-valine exporter YgaZH were also amplified by plasmid-based overexpression. The engineered E. coli W (ΔlacI ΔilvA) strain overexpressing the ilvBN(mut) , ilvCED, ygaZH, and lrp genes was able to produce an impressively high concentration of 60.7 g/L L-valine by fed-batch culture in 29.5 h, resulting in a high volumetric productivity of 2.06 g/L/h. The most notable finding is that there was no other byproduct produced during L-valine production. The results obtained in this study suggest that E. coli W can be a good alternative to Corynebacterium glutamicum and E. coli K-12, which have so far been the most efficient L-valine producer. Furthermore, it is expected that various bioproducts including other amino acids might be more efficiently produced by this revisited platform strain of E. coli.

  19. Cytosolic re-localization and optimization of valine synthesis and catabolism enables inseased isobutanol production with the yeast Saccharomyces cerevisiae

    PubMed Central

    2012-01-01

    Background The branched chain alcohol isobutanol exhibits superior physicochemical properties as an alternative biofuel. The yeast Saccharomyces cerevisiae naturally produces low amounts of isobutanol as a by-product during fermentations, resulting from the catabolism of valine. As S. cerevisiae is widely used in industrial applications and can easily be modified by genetic engineering, this microorganism is a promising host for the fermentative production of higher amounts of isobutanol. Results Isobutanol production could be improved by re-locating the valine biosynthesis enzymes Ilv2, Ilv5 and Ilv3 from the mitochondrial matrix into the cytosol. To prevent the import of the three enzymes into yeast mitochondria, N-terminally shortened Ilv2, Ilv5 and Ilv3 versions were constructed lacking their mitochondrial targeting sequences. SDS-PAGE and immunofluorescence analyses confirmed expression and re-localization of the truncated enzymes. Growth tests or enzyme assays confirmed enzymatic activities. Isobutanol production was only increased in the absence of valine and the simultaneous blockage of the mitochondrial valine synthesis pathway. Isobutanol production could be even more enhanced after adapting the codon usage of the truncated valine biosynthesis genes to the codon usage of highly expressed glycolytic genes. Finally, a suitable ketoisovalerate decarboxylase, Aro10, and alcohol dehydrogenase, Adh2, were selected and overexpressed. The highest isobutanol titer was 0.63 g/L at a yield of nearly 15 mg per g glucose. Conclusion A cytosolic isobutanol production pathway was successfully established in yeast by re-localization and optimization of mitochondrial valine synthesis enzymes together with overexpression of Aro10 decarboxylase and Adh2 alcohol dehydrogenase. Driving forces were generated by blocking competition with the mitochondrial valine pathway and by omitting valine from the fermentation medium. Additional deletion of pyruvate decarboxylase genes

  20. Comparative Genomics of Regulation of Fatty Acid and Branched-chain Amino Acid Utilization in Proteobacteria

    SciTech Connect

    Kazakov, Alexey E.; Rodionov, Dmitry A.; Arkin, Adam Paul; Dubchak, Inna; Gelfand, Mikhail S.; Alm, Eric

    2008-10-31

    Bacteria can use branched-chain amino acids (ILV, i.e. isoleucine, leucine, valine) and fatty acids (FA) as sole carbon and energy sources convering ILV into acetyl-CoA, propanoyl-CoA and propionyl-CoA, respectively. In this work, we used the comparative genomic approach to identify candidate transcriptional factors and DNA motifs that control ILV and FA utilization pathways in proteobacteria. The metabolic regulons were characterized based on the identification and comparison of candidate transcription factor binding sites in groups of phylogenetically related genomes. The reconstructed ILV/FA regulatory network demonstrates considerable variability and involves six transcriptional factors from the MerR, TetR and GntR families binding to eleven distinct DNA motifs. The ILV degradation genes in gamma- and beta-proteobacteria are mainly regulated by anovel regulator from the MerR family (e.g., LiuR in Pseudomonas aeruginosa) (40 species), in addition, the TetR-type regulator LiuQ was identified in some beta-proteobacteria (8 species). Besides the core set of ILV utilization genes, the LiuR regulon in some lineages is expanded to include genes from other metabolic pathways, such as the glyoxylate shunt and glutamate synthase in the Shewanella species. The FA degradation genes are controlled by four regulators including FadR in gamma-proteobacteria (34 species), PsrA in gamma- and beta-proteobacteria (45 species), FadP in beta-proteobacteria (14 species), and LiuR orthologs in alpha-proteobacteria (22 species). The remarkable variability of the regulatory systems associated with the FA degradation pathway is discussed from the functional and evolutionary points of view.

  1. (L)-Valine production with minimization of by-products' synthesis in Corynebacterium glutamicum and Brevibacterium flavum.

    PubMed

    Hou, Xiaohu; Chen, Xinde; Zhang, Yue; Qian, He; Zhang, Weiguo

    2012-12-01

    Corynebacterium glutamicum ATCC13032 and Brevibacterium flavum JV16 were engineered for L-valine production by over-expressing ilvEBN ( r ) C genes at 31 °C in 72 h fermentation. Different strategies were carried out to reduce the by-products' accumulation in L-valine fermentation and also to increase the availability of precursor for L-valine biosynthesis. The native promoter of ilvA of C. glutamicum was replaced with a weak promoter MPilvA (P-ilvAM1CG) to reduce the biosynthetic rate of L-isoleucine. Effect of different relative dissolved oxygen on L-valine production and by-products' formation was recorded, indicating that 15 % saturation may be the most appropriate relative dissolved oxygen for L-valine fermentation with almost no L-lactic acid and L-glutamate formed. To minimize L-alanine accumulation, alaT and/or avtA was inactivated in C. glutamicum and B. flavum, respectively. Compared to high concentration of L-alanine accumulated by alaT inactivated strains harboring ilvEBN ( r ) C genes, L-alanine concentration was reduced to 0.18 g/L by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ( r ) C, and 0.22 g/L by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ( r ) C. Meanwhile, L-valine production and conversion efficiency were enhanced to 31.15 g/L and 0.173 g/g by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ( r ) C, 38.82 g/L and 0.252 g/g by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ( r ) C. This study provides combined strategies to improve L-valine yield by minimization of by-products' production.

  2. Defective Transamination, a Mechanism for Resistance to Ketomycin in Escherichia coli

    PubMed Central

    Jackson, Julius H.; Umbarger, H. E.

    1973-01-01

    A spontaneous mutant of a derivative of Escherichia coli strain K-12 resistant to 50 μg of ketomycin per ml was selected. The mutant displayed a two- to threefold derepression of the isoleucine-valine biosynthetic enzymes and a reduced growth rate in minimal medium. The lesion was found to lie in the gene (ilvE) specifying transaminase B and resulted in an isoleucine limitation. The presence of exogenous isoleucine during growth in minimal medium restored normal phenotypic properties. The reduced transaminase B activity is responsible for the resistance to ketomycin. An unusual derepression of the acetohydroxy acid synthetase in response to an isoleucine limitation was noted. PMID:4597727

  3. Disposal Analysis of I-129 Bearing Waste Streams at the Intermediate Level Vault

    SciTech Connect

    Collard, L.B.

    2001-01-25

    This report examines the effects of new waste-specific sorption characteristics reported for I-129 bearing wastes on inventory limits in the Intermediate Level Vault (ILV). Inventory limits are described based on the revised performance assessment model using the waste-specific Kd's. Results are compared with inventory projections of waste streams for the next ten years.

  4. Human heart rate variability relation is unchanged during motion sickness

    NASA Technical Reports Server (NTRS)

    Mullen, T. J.; Berger, R. D.; Oman, C. M.; Cohen, R. J.

    1998-01-01

    In a study of 18 human subjects, we applied a new technique, estimation of the transfer function between instantaneous lung volume (ILV) and instantaneous heart rate (HR), to assess autonomic activity during motion sickness. Two control recordings of ILV and electrocardiogram (ECG) were made prior to the development of motion sickness. During the first, subjects were seated motionless, and during the second they were seated rotating sinusoidally about an earth vertical axis. Subjects then wore prism goggles that reverse the left-right visual field and performed manual tasks until they developed moderate motion sickness. Finally, ILV and ECG were recorded while subjects maintained a relatively constant level of sickness by intermittent eye closure during rotation with the goggles. Based on analyses of ILV to HR transfer functions from the three conditions, we were unable to demonstrate a change in autonomic control of heart rate due to rotation alone or due to motion sickness. These findings do not support the notion that moderate motion sickness is manifested as a generalized autonomic response.

  5. A dual-input nonlinear system analysis of autonomic modulation of heart rate

    NASA Technical Reports Server (NTRS)

    Chon, K. H.; Mullen, T. J.; Cohen, R. J.

    1996-01-01

    Linear analyses of fluctuations in heart rate and other hemodynamic variables have been used to elucidate cardiovascular regulatory mechanisms. The role of nonlinear contributions to fluctuations in hemodynamic variables has not been fully explored. This paper presents a nonlinear system analysis of the effect of fluctuations in instantaneous lung volume (ILV) and arterial blood pressure (ABP) on heart rate (HR) fluctuations. To successfully employ a nonlinear analysis based on the Laguerre expansion technique (LET), we introduce an efficient procedure for broadening the spectral content of the ILV and ABP inputs to the model by adding white noise. Results from computer simulations demonstrate the effectiveness of broadening the spectral band of input signals to obtain consistent and stable kernel estimates with the use of the LET. Without broadening the band of the ILV and ABP inputs, the LET did not provide stable kernel estimates. Moreover, we extend the LET to the case of multiple inputs in order to accommodate the analysis of the combined effect of ILV and ABP effect on heart rate. Analyzes of data based on the second-order Volterra-Wiener model reveal an important contribution of the second-order kernels to the description of the effect of lung volume and arterial blood pressure on heart rate. Furthermore, physiological effects of the autonomic blocking agents propranolol and atropine on changes in the first- and second-order kernels are also discussed.

  6. Platform engineering of Corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate.

    PubMed

    Buchholz, Jens; Schwentner, Andreas; Brunnenkan, Britta; Gabris, Christina; Grimm, Simon; Gerstmeir, Robert; Takors, Ralf; Eikmanns, Bernhard J; Blombach, Bastian

    2013-09-01

    Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a YP/S of 0.24 mol per mol of glucose and a QP of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

  7. Improvement of the redox balance increases L-valine production by Corynebacterium glutamicum under oxygen deprivation conditions.

    PubMed

    Hasegawa, Satoshi; Uematsu, Kimio; Natsuma, Yumi; Suda, Masako; Hiraga, Kazumi; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

    2012-02-01

    Production of L-valine under oxygen deprivation conditions by Corynebacterium glutamicum lacking the lactate dehydrogenase gene ldhA and overexpressing the L-valine biosynthesis genes ilvBNCDE was repressed. This was attributed to imbalanced cofactor production and consumption in the overall L-valine synthesis pathway: two moles of NADH was generated and two moles of NADPH was consumed per mole of L-valine produced from one mole of glucose. In order to solve this cofactor imbalance, the coenzyme requirement for L-valine synthesis was converted from NADPH to NADH via modification of acetohydroxy acid isomeroreductase encoded by ilvC and introduction of Lysinibacillus sphaericus leucine dehydrogenase in place of endogenous transaminase B, encoded by ilvE. The intracellular NADH/NAD(+) ratio significantly decreased, and glucose consumption and L-valine production drastically improved. Moreover, L-valine yield increased and succinate formation decreased concomitantly with the decreased intracellular redox state. These observations suggest that the intracellular NADH/NAD(+) ratio, i.e., reoxidation of NADH, is the primary rate-limiting factor for L-valine production under oxygen deprivation conditions. The L-valine productivity and yield were even better and by-products derived from pyruvate further decreased as a result of a feedback resistance-inducing mutation in the acetohydroxy acid synthase encoded by ilvBN. The resultant strain produced 1,470 mM L-valine after 24 h with a yield of 0.63 mol mol of glucose(-1), and the L-valine productivity reached 1,940 mM after 48 h.

  8. Wavemaker theories for acoustic-gravity waves over a finite depth

    NASA Astrophysics Data System (ADS)

    Tian, Miao; Kadri, Usama

    2016-04-01

    Acoustic-gravity waves (hereafter AGWs) in ocean have received much interest recently, mainly with respect to early detection of tsunamis as they travel at near the speed of sound in water which makes them ideal candidates for early detection of tsunamis. While the generation mechanisms of AGWs have been studied from the perspective of vertical oscillations of seafloor (Yamamoto, 1982; Stiassnie, 2010) and triad wave-wave interaction (Longuet-Higgins 1950; Kadri and Stiassnie 2013; Kadri and Akylas 2016), in the current study we are interested in their generation by wave-structure interaction with possible application to the energy sector. Here, we develop two wavemaker theories to analyze different wave modes generated by impermeable (the classic Havelock's theory) and porous (porous wavemaker theory) plates in weakly compressible fluids. Slight modification has been made to the porous theory so that, unlike the previous theory (Chwang, 1983), the new solution depends on the geometry of the plate. The expressions for three different types of plates (piston, flap, delta-function) are introduced. Analytical solutions are also derived for the potential amplitude of the gravity, evanescent, and acoustic-gravity waves, as well as the surface elevation, velocity distribution, and pressure for AGWs. Both theories reduce to previous results for incompressible flow when the compressibility is negligible. We also show numerical examples for AGW generated in a wave flume as well as in deep ocean. Our current study sets the theoretical background towards remote sensing by AGWs, for optimized deep ocean wave-power harnessing, among others. References Chwang, A.T. 1983 A porous-wavemaker theory. Journal of Fluid Mechanics, 132, 395- 406. Kadri, U., Stiassnie, M. 2013 Generation of an acoustic-gravity wave by two gravity waves, and their subsequent mutual interaction. J. Fluid Mech. 735, R6. Kadri U., Akylas T.R. 2016 On resonant triad interactions of acoustic-gravity waves. J

  9. A NMR experiment for simultaneous correlations of valine and leucine/isoleucine methyls with carbonyl chemical shifts in proteins.

    PubMed

    Tugarinov, Vitali; Venditti, Vincenzo; Marius Clore, G

    2014-01-01

    A methyl-detected 'out-and-back' NMR experiment for obtaining simultaneous correlations of methyl resonances of valine and isoleucine/leucine residues with backbone carbonyl chemical shifts, SIM-HMCM(CGCBCA)CO, is described. The developed pulse-scheme serves the purpose of convenience in recording a single data set for all Ile(δ1), Leu(δ) and Val(γ) (ILV) methyl positions instead of acquiring two separate spectra selective for valine or leucine/isoleucine residues. The SIM-HMCM(CGCBCA)CO experiment can be used for ILV methyl assignments in moderately sized protein systems (up to ~100 kDa) where the backbone chemical shifts of (13)C(α), (13)Cβ and (13)CO are known from prior NMR studies and where some losses in sensitivity can be tolerated for the sake of an overall reduction in NMR acquisition time.

  10. Selective Methyl Labeling of Eukaryotic Membrane Proteins Using Cell-Free Expression

    PubMed Central

    2015-01-01

    Structural characterization of membrane proteins and other large proteins with NMR relies increasingly on perdeuteration combined with incorporation of specifically protonated amino acid moieties, such as methyl groups of isoleucines, valines, or leucines. The resulting proton dilution reduces dipolar broadening producing sharper resonance lines, ameliorates spectral crowding, and enables measuring of crucial distances between and to methyl groups. While incorporation of specific methyl labeling is now well established for bacterial expression using suitable precursors, corresponding methods are still lacking for cell-free expression, which is often the only choice for producing labeled eukaryotic membrane proteins in mg quantities. Here we show that we can express methyl-labeled human integral membrane proteins cost-effectively by cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to channel ILV precursors into the eukaryotic proteins. PMID:24937763

  11. Determination of the dissociation constant of valine from acetohydroxy acid synthase by equilibrium partition in an aqueous two-phase system.

    PubMed

    Engel, S; Vyazmensky, M; Barak, Z; Chipman, D M; Merchuk, J C

    2000-06-23

    An aqueous polyethylene glycol/salt two-phase system was used to estimate the dissociation constant, K(dis), of the Escherichia coli isoenzyme AHAS III regulatory subunit, ilvH protein, from the feedback inhibitor valine. The amounts of the bound and free radioactive valine in the system were determined. A Scatchard plot of the data revealed a 1:1 valine-protein binding ratio and K(dis) of 133+/-14 microM. The protein did not bind leucine, and the ilvH protein isolated from a valine resistant mutant showed no valine binding. This method is very simple, rapid and requires only a small amounts of protein compared to the presently used equilibrium dialysis method.

  12. Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha

    PubMed Central

    Ushimaru, Kazunori; Mizuno, Shoji

    2015-01-01

    Recombinant Ralstonia eutropha strain PHB−4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB−4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB−4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone. PMID:25645560

  13. GTP dysregulation in Bacillus subtilis cells lacking (p)ppGpp results in phenotypic amino acid auxotrophy and failure to adapt to nutrient downshift and regulate biosynthesis genes.

    PubMed

    Kriel, Allison; Brinsmade, Shaun R; Tse, Jessica L; Tehranchi, Ashley K; Bittner, Alycia N; Sonenshein, Abraham L; Wang, Jue D

    2014-01-01

    The nucleotide (p)ppGpp inhibits GTP biosynthesis in the Gram-positive bacterium Bacillus subtilis. Here we examined how this regulation allows cells to grow in the absence of amino acids. We showed that B. subtilis cells lacking (p)ppGpp, due to either deletions or point mutations in all three (p)ppGpp synthetase genes, yjbM, ywaC, and relA, strongly require supplementation of leucine, isoleucine, valine, methionine, and threonine and modestly require three additional amino acids. This polyauxotrophy is rescued by reducing GTP levels. Reduction of GTP levels activates transcription of genes responsible for the biosynthesis of the five strongly required amino acids by inactivating the transcription factor CodY, which represses the ybgE, ilvD, ilvBHC-leuABCD, ilvA, ywaA, and hom-thrCB operons, and by a CodY-independent activation of transcription of the ilvA, ywaA, hom-thrCB, and metE operons. Interestingly, providing the eight required amino acids does not allow for colony formation of (p)ppGpp(0) cells when transitioning from amino acid-replete medium to amino acid-limiting medium, and we found that this is due to an additional role that (p)ppGpp plays in protecting cells during nutrient downshifts. We conclude that (p)ppGpp allows adaptation to amino acid limitation by a combined effect of preventing death during metabolic transitions and sustaining growth by activating amino acid biosynthesis. This ability of (p)ppGpp to integrate a general stress response with a targeted reprogramming of gene regulation allows appropriate adaptation and is likely conserved among diverse bacteria.

  14. Enhanced production of branched-chain amino acids by Gluconacetobacter europaeus with a specific regional deletion in a leucine responsive regulator.

    PubMed

    Akasaka, Naoki; Ishii, Yuri; Hidese, Ryota; Sakoda, Hisao; Fujiwara, Shinsuke

    2014-12-01

    Vinegar with increased amounts of branched-chain amino acids (BCAAs; valine, leucine and isoleucine) is favorable for human health as BCAAs decrease diet-induced obesity and hyperglycemia. To construct Gluconacetobacter europaeus which produces BCAAs, leucine responsive regulator (GeLrp) is focused and two Gelrp mutants were constructed. Wild-type KGMA0119 didn't produce significant amount of valine (0.13 mM) and leucine (0 mM) and strain KGMA7110 which lacks complete Gelrp accumulated valine (0.48 mM) and leucine (0.11 mM) but showed impaired growth, and it was fully restored in the presence of essential amino acids. Strain KGMA7203 was then constructed with a nonsense mutation at codon Trp132 in the Gelrp, which leads a specific deletion at an estimated ligand-sensing region in the C-terminal domain. KGMA7203 produced greater quantities of valine (0.80 mM) and leucine (0.26 mM) and showed the same growth characteristics as KGMA0119. mRNA levels of BCAAs biosynthesis genes (ilvI and ilvC) and probable BCAAs efflux pump (leuE) were determined by quantitative reverse-transcription PCR. Expression rates of ilvI and ilvC in the two Gelrp disruptants were greater than those in KGMA0119. leuE was highly expressed in KGMA7110 only, suggesting that the accumulation in KGMA7110 culture was caused by increased expression of the biosynthesis genes and abnormal enhanced export of amino acids resulting in impaired cell growth. In contrast, KGMA7203 would achieve the high level production through enhanced expression of the biosynthesis genes without enhancing that for the efflux pump. KGMA7203 was considered advantageous for production of vinegar with higher amounts of valine and leucine.

  15. Role for Free Isoleucine or Glycyl-Leucine in the Repression of Threonine Deaminase in Escherichia coli

    PubMed Central

    Wasmuth, John J.; Umbarger, H. E.

    1974-01-01

    In Escherichia coli, the three branched-chain amino acid activating enzymes appear to be essential for multivalent repression of the isoleucine- and valine-forming enzymes. The results of experiments with a mutant, strain CU18, having an altered threonine deaminase, indicate that free isoleucine and some form of threonine deaminase (the product of the ilvA gene) are also involved in multivalent repression. This strain exhibits abnormally high derepressibility but normal repressibility of its ilv gene products, and its threonine deaminase is inhibited only by high concentrations of isoleucine. In strain CU18, the isoleucine analogue, thiaisoleucine, is incapable of replacing isoleucine in the multivalent repression of the ilv genes, whereas the analogue can fully replace the natural amino acid in repression in other strains examined. The dipeptide, glycyl-leucine, which, like isoleucine, is a heterotropic negative effector of threonine deaminase but is not a substrate for isoleucyl-transfer ribonucleic acid synthetase, can completely prevent the accumulation of threonine deaminase-forming potential during isoleucine starvation in strains with normal threonine deaminases. It can not, however, prevent such accumulation in strain CU18 or in other strains in which threonine deaminase is insensitive to any concentration of isoleucine. PMID:4587610

  16. Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes.

    PubMed

    Lee, Won-Heong; Seo, Seung-Oh; Bae, Yi-Hyun; Nan, Hong; Jin, Yong-Su; Seo, Jin-Ho

    2012-11-01

    Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93 mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151 mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.

  17. Enhanced valine production in Corynebacterium glutamicum with defective H+-ATPase and C-terminal truncated acetohydroxyacid synthase.

    PubMed

    Wada, Masaru; Hijikata, Nowaki; Aoki, Ryo; Takesue, Nobuchika; Yokota, Atsushi

    2008-11-01

    We have reported increased glutamate production by a mutant of Corynebacterium glutamicum ATCC14067 (strain F172-8) with reduced H(+)-ATPase activity under biotin-limiting culture conditions (Aoki et al. Biosci. Biotechnol. Biochem., 69, 1466-1472 (2005)). In the present study, we examined valine production by an H(+)-ATPase-defective mutant of C. glutamicum. Using the double-crossover chromosome replacement technique, we constructed a newly defined H(+)-ATPase-defective mutant from ATCC13032. After transforming the new strain (A-1) with a C-terminal truncation of acetohydroxyacid synthase gene (ilvBN), valine production increased from 21.7 mM for the wild-type strain to 46.7 mM for the A-1 in shaking flask cultures with 555 mM glucose. Increased production of the valine intermediate acetoin was also observed in A-1, and was reduced by inserting acetohydroxyacid isomeroreductase gene (ilvC) into the ilvBN plasmid. After transformation with this new construct, valine production increased from 38.3 mM for the wild-type strain to 95.7 mM for A-1 strain. To the best of our knowledge, this is the first report indicating that an H(+)-ATPase-defective mutant of C. glutamicum is capable of valine production. Our combined results with glutamate and valine suggest that the H(+)-ATPase defect is also effective in the fermentative production of other practical compounds.

  18. System identification of closed-loop cardiovascular control: effects of posture and autonomic blockade

    NASA Technical Reports Server (NTRS)

    Mullen, T. J.; Appel, M. L.; Mukkamala, R.; Mathias, J. M.; Cohen, R. J.

    1997-01-01

    We applied system identification to the analysis of fluctuations in heart rate (HR), arterial blood pressure (ABP), and instantaneous lung volume (ILV) to characterize quantitatively the physiological mechanisms responsible for the couplings between these variables. We characterized two autonomically mediated coupling mechanisms [the heart rate baroreflex (HR baroreflex) and respiratory sinus arrhythmia (ILV-HR)] and two mechanically mediated coupling mechanisms [the blood pressure wavelet generated with each cardiac contraction (circulatory mechanics) and the direct mechanical effects of respiration on blood pressure (ILV-->ABP)]. We evaluated the method in humans studied in the supine and standing postures under control conditions and under conditions of beta-sympathetic and parasympathetic pharmacological blockades. Combined beta-sympathetic and parasympathetic blockade abolished the autonomically mediated couplings while preserving the mechanically mediated coupling. Selective autonomic blockade and postural changes also altered the couplings in a manner consistent with known physiological mechanisms. System identification is an "inverse-modeling" technique that provides a means for creating a closed-loop model of cardiovascular regulation for an individual subject without altering the underlying physiological control mechanisms.

  19. Clusters of isoleucine, leucine, and valine side chains define cores of stability in high-energy states of globular proteins: Sequence determinants of structure and stability.

    PubMed

    Kathuria, Sagar V; Chan, Yvonne H; Nobrega, R Paul; Özen, Ayşegül; Matthews, C Robert

    2016-03-01

    Measurements of protection against exchange of main chain amide hydrogens (NH) with solvent hydrogens in globular proteins have provided remarkable insights into the structures of rare high-energy states that populate their folding free-energy surfaces. Lacking, however, has been a unifying theory that rationalizes these high-energy states in terms of the structures and sequences of their resident proteins. The Branched Aliphatic Side Chain (BASiC) hypothesis has been developed to explain the observed patterns of protection in a pair of TIM barrel proteins. This hypothesis supposes that the side chains of isoleucine, leucine, and valine (ILV) residues often form large hydrophobic clusters that very effectively impede the penetration of water to their underlying hydrogen bond networks and, thereby, enhance the protection against solvent exchange. The linkage between the secondary and tertiary structures enables these ILV clusters to serve as cores of stability in high-energy partially folded states. Statistically significant correlations between the locations of large ILV clusters in native conformations and strong protection against exchange for a variety of motifs reported in the literature support the generality of the BASiC hypothesis. The results also illustrate the necessity to elaborate this simple hypothesis to account for the roles of adjacent hydrocarbon moieties in defining stability cores of partially folded states along folding reaction coordinates.

  20. Recharge Data for Hawaii Island

    DOE Data Explorer

    Nicole Lautze

    2015-01-01

    Recharge data for Hawaii Island in shapefile format. The data are from the following sources: Whittier, R.B and A.I. El-Kadi. 2014. Human Health and Environmental Risk Ranking of On-Site Sewage Disposal systems for the Hawaiian Islands of Kauai, Molokai, Maui, and Hawaii – Final, Prepared for Hawaii Dept. of Health, Safe Drinking Water Branch by the University of Hawaii, Dept. of Geology and Geophysics. Oki, D. S. 1999. Geohydrology and Numerical Simulation of the Ground-Water Flow System of Kona, Island of Hawaii. U.S. Water-Resources Investigation Report: 99-4073. Oki, D. S. 2002. Reassessment of Ground-water Recharge and Simulated Ground-Water Availability for the Hawi Area of North Kohala, Hawaii. U.S. Geological Survey Water-Resources Investigation report 02-4006.

  1. Assessment of groundwater quality with respect to fluoride.

    PubMed

    Salve, P R; Maurya, A; Kumbhare, P S; Ramteke, D S; Wate, S R

    2008-09-01

    The study was conducted in the summer season (April-May 2007). The fluoride concentration along with physico-chemical parameters in ground water samples was determined in various villages of Kadi tehsil at Mehsana district of Gujarat state (India), since in most of the villages it is the only source of drinking water. The fluoride concentrations in these villages varied from 0.94 to 2.81 mg/L (1.37+/-0.56) with highest fluoride level at Visalpur (2.08 mg/L) and lowest at Adaraj (0.91 mg/L). There was found a positive correlation of pH with fluoride and a negative relationship of fluoride with bicarbonate which is generally observed in deep ground water.

  2. Assessment of the state of the gibel carp Carassius auratus gibelio in the Amur River Basin: heavy-metal and arsenic concentrations and histopathology of internal organs.

    PubMed

    Syasina, Iraida G; Khlopova, Anna V; Chukhlebova, Lyubov M

    2012-04-01

    This study describes the concentrations of heavy metals and arsenic (As) and the basic histopathological changes in the internal organs of gibel carp Carassius auratus gibelio from five sites of the Amur River basin. Gibel carp from Sindinskaya Passage had the highest liver concentrations of zinc (Zn) (31.95 ± 13.443), copper (Cu) (12.52 ± 5.746), manganese (9.22 ± 8.121), and cadmium (0.37 ± 0.660 mg/kg wet weight [ww]) compared with fish captured from the Bol'shoi Ussuriiskii Island area and Kadi Lake; however, concentrations of nickel (0.22 ± 0.156 mg/kg ww) were not significantly different, and concentrations of lead (0.19 ± 0.121 mg/kg ww) were higher than those in fish from Kadi Lake. Mean concentrations of metals and As in muscles were lower than Russia's recommended limits for food products; however, concentrations of Cu, Zn, and Hg in individual fish were greater than the limit. Kidney disease was detected in 100% of sampled carp. Kidney disease was characterized by the formation of numerous granulomas in kidney tissues between the renal tubules. The degree of granulomatosis varied among sites. Granulomatous kidney disease in gibel carp is widespread in many reservoirs of the lower Amur River basin. The following histopathological changes were detected in liver: vacuolization of hepatocytes, hypertrophy of multiple hepatocytes, binuclearity, presence of numerous irregularly shaped nuclei in hepatocytes, karyopyknosis, diffuse necrosis of hepatocytes (in some cases focal), and edema. Necrotic changes in hepatocytes, which are important indices of the toxic effect of pollutants, were found in the majority of investigated carp from the lower Amur River basin. Some fish had simultaneous pathological alterations in multiple organs.

  3. A tightly packed hydrophobic cluster directs the formation of an off-pathway sub-millisecond folding intermediate in the alpha subunit of tryptophan synthase, a TIM barrel protein.

    PubMed

    Wu, Ying; Vadrevu, Ramakrishna; Kathuria, Sagar; Yang, Xiaoyan; Matthews, C Robert

    2007-03-01

    Protein misfolding is now recognized as playing a crucial role in both normal and pathogenic folding reactions. An interesting example of misfolding at the earliest state of a natural folding reaction is provided by the alpha-subunit of tryptophan synthase, a (beta/alpha)(8) TIM barrel protein. The molecular basis for the formation of this off-pathway misfolded intermediate, I(BP), and a subsequent on-pathway intermediate, I1, was probed by mutational analysis of 20 branched aliphatic side-chains distributed throughout the sequence. The elimination of I(BP) and the substantial destabilization of I1 by replacement of a selective set of the isoleucine, leucine or valine residues (ILV) with alanine in a large ILV cluster external-to-the-barrel and spanning the N and C termini (cluster 2) implies tight-packing at most sites in both intermediates. Differential effects on I(BP) and I1 for replacements in alpha3, beta4 and alpha8 at the boundaries of cluster 2 suggest that their incorporation into I1 but not I(BP) reflects non-native folds at the edges of the crucial (beta/alpha)(1-2)beta(3) core in I(BP). The retention of I(BP) and the smaller and consistent destabilization of both I(BP) and I1 by similar replacements in an internal-to-the-barrel ILV cluster (cluster 1) and a second external-to-the-barrel ILV cluster (cluster 3) imply molten globule-like packing. The tight packing inferred, in part, for I(BP) or for all of I1 in cluster 2, but not in clusters 1 and 3, may reflect the larger size of cluster 2 and/or the enhanced number of isoleucine, leucine and valine self-contacts in and between contiguous elements of secondary structure. Tightly packed ILV-dominated hydrophobic clusters could serve as an important driving force for the earliest events in the folding and misfolding of the TIM barrel and other members of the (beta/alpha)(n) class of proteins.

  4. [Construction of the new Escherichia coli K-12 wild-type strain with improved growth characteristics for application in metabolic engineering].

    PubMed

    Biriukova, I V; Krylov, A A; Kiseleva, E M; Minaeva, N I; Mashko, S V

    2010-03-01

    MG1655 of Escherichia coli K-12 is frequently used in metabolic engineering as the wild-type strain. However, its two mutations, ilvG and rph-1 provide a negative effect on culture growth. The "polar effect" of rph-1 decreases the level of pyrE expression, causing partial auxotrophy for pyrimidines. Mutation ilvG leading to the appearance of Val(S) phenotype causes retardation of cell growth rate on media containing amino acids. In this work, the substitution of two loci in the genome of MG1655 with the recovery of the wild-type phenotype was accomplished. Gene rph(wt) from the chromosome of E. coli TG1 was marked via Red-dependent integration of DNA fragment carrying lambda attL-Cm(R)-lambda attR and transduced with phage P1 into MG1655; later, the Cm(R) marker was removed with the use of lambda Xis/Int recombinase. Parallel to this procedure, a spontaneous Val(R) mutant of E. coli MG1655 yielding colonies of maximal size on M9 medium with glucose in the presence of Val (50 microg/ml) was isolated. It was shown that a nucleotide deletion in the isolated Val(R) strain had been generated in the region of the identified E. coli K-12 ilvG mutation, which led to the recovery of the reading frame and active protein synthesis. This mutation named ilvG-15, which is the only reason for the Val(R) phenotype in the obtained strain, was transferred to MG1655-rph(wt) using cotransduction, by analogy to the transfer of rph(wt). Evaluation of rates of aerobically growing cells (microm, hour(-1)) on M9 medium with glucose produced the following values: 0.56, 0.69, and 0.73 for strains MG1655, MG1655-rph(wt), and MG1655-(rph(wt), ilvG-15), respectively.

  5. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16.

    PubMed

    Lu, Jingnan; Brigham, Christopher J; Plassmeier, Jens K; Sinskey, Anthony J

    2015-01-01

    2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by L-valine (IC50=1.2 mM), L-isoleucine (IC50=2.3 mM), and L-leucine (IC50=5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (KM=10.5 μM) and is highly selective towards 2-ketobutyrate (R=140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for

  6. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16

    SciTech Connect

    Lu, JN; Brigham, CJ; Plassmeier, JK; Sinskey, AJ

    2014-08-01

    2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by l-valine (IC50 = 1.2 mM), l-isoleucine (IC50 = 2.3 mM), and l-leucine (IC50 = 5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (K-M = 10.5 mu M) and is highly selective towards 2-ketobutyrate (R = 140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2

  7. Enhanced biosynthetically directed fractional carbon-13 enrichment of proteins for backbone NMR assignments.

    PubMed

    Wenrich, Broc R; Sonstrom, Reilly E; Gupta, Riju A; Rovnyak, David

    2015-11-01

    Routes to carbon-13 enrichment of bacterially expressed proteins include achieving uniform or positionally selective (e.g. ILV-Me, or (13)C', etc.) enrichment. We consider the potential for biosynthetically directed fractional enrichment (e.g. carbon-13 incorporation in the protein less than 100%) for performing routine n-(D)dimensional NMR spectroscopy of proteins. First, we demonstrate an approach to fractional isotope addition where the initial growth media containing natural abundance glucose is replenished at induction with a small amount (e.g. 10%(w/w)u-(13)C-glucose) of enriched nutrient. The approach considered here is to add 10% (e.g. 200mg for a 2g/L culture) u-(13)C-glucose at the induction time (OD600=0.8), resulting in a protein with enhanced (13)C incorporation that gives almost the same NMR signal levels as an exact 20% (13)C sample. Second, whereas fractional enrichment is used for obtaining stereospecific methyl assignments, we find that (13)C incorporation levels no greater than 20%(w/w) yield (13)C and (13)C-(13)C spin pair incorporation sufficient to conduct typical 3D-bioNMR backbone experiments on moderate instrumentation (600 MHz, RT probe). Typical 3D-bioNMR experiments of a fractionally enriched protein yield expected backbone connectivities, and did not show amino acid biases in this work, with one exception. When adding 10% u-(13)C glucose to expression media at induction, there is poor preservation of (13)Cα-(13)Cβ spin pairs in the amino acids ILV, leading to the absence of Cβ signals in HNCACB spectra for ILV, a potentially useful editing effect. Enhanced fractional carbon-13 enrichment provides lower-cost routes to high throughput protein NMR studies, and makes modern protein NMR more cost-accessible.

  8. Class E compartments form in response to ESCRT dysfunction in yeast due to hyperactivity of the Vps21 Rab GTPase.

    PubMed

    Russell, Matthew R G; Shideler, Tess; Nickerson, Daniel P; West, Matt; Odorizzi, Greg

    2012-11-01

    The endosomal sorting complexes required for transport (ESCRTs) mediate the budding of intralumenal vesicles (ILVs) at late endosomes. ESCRT dysfunction causes drastic changes in endosome morphology, which are manifested in Saccharomyces cerevisiae by the formation of aberrant endosomes known as class E compartments. Except for the absence of ILVs, the mechanistic basis for class E compartment biogenesis is unknown. We used electron microscopy to examine endosomal morphology in response to transient ESCRT inactivation and recovery in yeast expressing the temperature-sensitive mutant vps4(ts) allele. Our results show class E compartments accumulate fourfold the amount of membrane normally present at multivesicular bodies and that multivesicular bodies can form directly from class E compartments upon recovery of ESCRT function. We found class E compartment formation requires Vps21, which is orthologous to the Rab5A GTPase in metazoans that promotes fusion of endocytic vesicles with early endosomes and homotypic fusion of early endosomes with one another. We also determined that class E compartments accumulate GTP-bound Vps21 and its effector, the class C core vacuole/endosome tethering (CORVET). Ypt7, the yeast ortholog of Rab7 that in metazoans promotes fusion of late endosomes with lysosomes, also accumulates at class E compartments but without its effector, the homotypic fusion and protein sorting (HOPS), signifying that Ypt7 at class E compartments is dysfunctional. These results suggest that failure to complete Rab5-Rab7 conversion is a consequence of ESCRT dysfunction, which results in Vps21 hyperactivity that drives the class E compartment morphology. Indeed, genetic disruption of Rab conversion without ESCRT dysfunction autonomously drives the class E compartment morphology without blocking ILV budding.

  9. Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain.

    PubMed

    Miyanoiri, Yohei; Ishida, Yojiro; Takeda, Mitsuhiro; Terauchi, Tsutomu; Inouye, Masayori; Kainosho, Masatsune

    2016-06-01

    We recently developed a practical protocol for preparing proteins bearing stereo-selectively (13)C-methyl labeled leucines and valines, instead of the commonly used (13)C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

  10. I-129 Desorption from SRS Water Treatment Media from the Effluent Treatment Facility and the F-Area Groundwater Treatment Facility

    SciTech Connect

    Kaplan, D.I.

    2001-01-26

    The objective of this study was to quantify I-129 desorption of four waste materials and to provide reasonably conservative Kd values for modeling efforts. Since the ILV PA considers dose thousands of years in the future, additional attention was directed at evaluating how I-129 desorption changed as a function of time. The scope of this work involved evaluating four waste materials (F-WTU Dowex 21K, F-WTU Sludge, ETF Carbon, and ETF GT-73) under two aqueous conditions (acid rain and cement simulants) by two different experimental protocols (static batch and dynamic flow column experiments).

  11. Genes involved in protein metabolism of the probiotic lactic acid bacterium Lactobacillus delbrueckii UFV H2b20.

    PubMed

    Do Carmo, A P; da Silva, D F; De Oliveira, M N V; Borges, A C; De Carvalho, A F; De Moraes, C A

    2011-09-01

    A basic requirement for the prediction of the potential use of lactic acid bacteria (LAB) in the dairy industry is the identification of specific genes involved in flavour-forming pathways. The probiotic Lactobacillus delbrueckii UFV H2b20 was submitted to a genetic characterisation and phylogenetic analysis of genes involved in protein catabolism. Eight genes belonging to this system were identified, which possess a closely phylogenetic relationship to NCFM strains representative, as it was demonstrated for oppC and oppBII, encoding oligopeptide transport system components. PepC, PepN, and PepX might be essential for growth of LAB, probiotic or not, since the correspondent genes are always present, including in L. delbrueckii UFV H2b20 genome. For pepX gene, a probable link between carbohydrate catabolism and PepX expression may exists, where it is regulated by PepR1/CcpA-like, a common feature between Lactobacillus strains and also in L. delbrueckii UFV H2b20. The well conserved evolutionary history of the ilvE gene is evidence that the pathways leading to branched-chain amino acid degradation, such as isoleucine and valine, are similar among L. delbrueckii subsp. bulgaricus strains and L. delbrueckii UFV H2b20. Thus, the involvement of succinate in flavour formation can be attributed to IlvE activity. The presence of aminopeptidase G in L. delbrueckii UFV H2b20 genome, which is absent in several strains, might improve the proteolytic activity and effectiveness. The nucleotide sequence encoding PepG revealed that it is a cysteine endopeptidase, belonging to Peptidase C1 superfamily; sequence analysis showed 99% identity with L. delbrueckii subsp. bulgaricus ATCC 11842 pepG, whereas protein sequence analysis revealed 100% similarity with PepG from the same organism. The present study proposes a schematic model to explain how the proteolytic system of the probiotic L. delbrueckii UFV H2b20 works, based on the components identified so far.

  12. Reconnaissance exploration geochemistry in the central Brooks Range, northern Alaska: Implications for exploration of sediment-hosted zinc-lead-silver deposits

    USGS Publications Warehouse

    Kelley, K.D.; Kelley, D.L.

    1992-01-01

    A reconnaissance geochemical survey was conducted in the southern Killik River quadrangle, central Brooks Range, northern Alaska. The Brooks Range lies within the zone of continuous permafrost which may partially inhibit chemical weathering and oxidation. The minus 30-mesh and nonmagnetic heavy-mineral concentrate fractions of sediment samples were chosen as the sample media for the survey so that mechanical rather than chemical dispersion patterns would be enhanced. A total of 263 sites were sampled within the southern half of the Killik River quadrangle at an average sample density of approximately one sample per 12 km2. All samples were submitted for multi-element analyses. In the western and central Brooks Range, several known sediment-hosted Zn-Pb-Ag(-Ba) deposits occur within a belt of Paleozoic rocks of the Endicott Mountains allochthon. Exploration for this type of deposit in the Brook Range is difficult, due to the inherently high background values for Ba, Zn and Pb in shale and the common occurrence of metamorphic quartz-calcite veins, many of which contain traces of sulfide minerals. Stream sediments derived from these sources produce numerous geochemical anomalies which are not necessarily associated with significant mineralization. R-mode factor analysis provides a means of distinguishing between element associations related to lithology and those related to possible mineralization. Factor analysis applied to the multi-element data from the southern Killik River quadrangle resulted in the discovery of two additional Zn-Pb-Ag mineral occurrences of considerable areal extent which are 80-100 km east of any previously known deposit. These have been informally named the Kady and Vidlee. Several lithogeochemical element associations, or factors, and three factors which represent sulfide mineralization were identified: Ag-Pb-Zn (galena and sphalerite) and Fe-Ni-Co-Cu (pyrite ?? chalcopyrite) in the concentrate samples and Cd-Zn-Pb-As-Mn in the sediment

  13. An Unexpected Route to an Essential Cofactor: Escherichia coli Relies on Threonine for Thiamine Biosynthesis

    PubMed Central

    Bazurto, Jannell V.; Farley, Kristen R.

    2016-01-01

    ABSTRACT Metabolism consists of biochemical reactions that are combined to generate a robust metabolic network that can respond to perturbations and also adapt to changing environmental conditions. Escherichia coli and Salmonella enterica are closely related enterobacteria that share metabolic components, pathway structures, and regulatory strategies. The synthesis of thiamine in S. enterica has been used to define a node of the metabolic network by analyzing alternative inputs to thiamine synthesis from diverse metabolic pathways. To assess the conservation of metabolic networks in organisms with highly conserved components, metabolic contributions to thiamine synthesis in E. coli were investigated. Unexpectedly, we found that, unlike S. enterica, E. coli does not use the phosphoribosylpyrophosphate (PRPP) amidotransferase (PurF) as the primary enzyme for synthesis of phosphoribosylamine (PRA). In fact, our data showed that up to 50% of the PRA used by E. coli to make thiamine requires the activities of threonine dehydratase (IlvA) and anthranilate synthase component II (TrpD). Significantly, the IlvA- and TrpD-dependent pathway to PRA functions in S. enterica only in the absence of a functional reactive intermediate deaminase (RidA) enzyme, bringing into focus how these closely related bacteria have distinct metabolic networks. PMID:26733068

  14. Proteins involved in wine aroma compounds metabolism by a Saccharomyces cerevisiae flor-velum yeast strain grown in two conditions.

    PubMed

    Moreno-García, Jaime; García-Martínez, Teresa; Millán, M Carmen; Mauricio, Juan Carlos; Moreno, Juan

    2015-10-01

    A proteomic and exometabolomic study was conducted on Saccharomyces cerevisiae flor yeast strain growing under biofilm formation condition (BFC) with ethanol and glycerol as carbon sources and results were compared with those obtained under no biofilm formation condition (NBFC) containing glucose as carbon source. By using modern techniques, OFFGEL fractionator and LTQ-Orbitrap for proteome and SBSE-TD-GC-MS for metabolite analysis, we quantified 84 proteins including 33 directly involved in the metabolism of glycerol, ethanol and 17 aroma compounds. Contents in acetaldehyde, acetic acid, decanoic acid, 1,1-diethoxyethane, benzaldehyde and 2-phenethyl acetate, changed above their odor thresholds under BFC, and those of decanoic acid, ethyl octanoate, ethyl decanoate and isoamyl acetate under NBFC. Of the twenty proteins involved in the metabolism of ethanol, acetaldehyde, acetoin, 2,3-butanediol, 1,1-diethoxyethane, benzaldehyde, organic acids and ethyl esters, only Adh2p, Ald4p, Cys4p, Fas3p, Met2p and Plb1p were detected under BFC and as many Acs2p, Ald3p, Cem1p, Ilv2p, Ilv6p and Pox1p, only under NBFC. Of the eight proteins involved in glycerol metabolism, Gut2p was detected only under BFC while Pgs1p and Rhr2p were under NBFC. Finally, of the five proteins involved in the metabolism of higher alcohols, Thi3p was present under BFC, and Aro8p and Bat2p were under NBFC.

  15. Characterization of acetohydroxyacid synthase I from Escherichia coli K-12 and identification of its inhibitors.

    PubMed

    Pham, Ngoc Chien; Moon, Ji-Young; Cho, Jun-Haeng; Lee, Soo-Jae; Park, Joon-Shik; Kim, Dong-Eun; Park, Yoonkyung; Yoon, Moon-Young

    2010-01-01

    The first step in branched-chain amino acid biosynthesis is catalyzed by acetohydroxyacid synthase (EC 2.2.1.6). This reaction involves decarboxylation of pyruvate followed by condensation with either an additional pyruvate molecule or with 2-oxobutyrate. The enzyme requires three cofactors, thiamine diphosphate (ThDP), a divalent ion, and flavin adenine dinucleotide (FAD). Escherichia coli contains three active isoenzymes, and acetohydroxyacid synthase I (AHAS I) large subunit is encoded by the ilvB gene. In this study, the ilvB gene from E. coli K-12 was cloned into expression vector pETDuet-1, and was expressed in E. coli BL21 (DH3). The purified protein was identified on a 12% SDS-PAGE gel as a single band with a mass of 65 kDa. The optimum temperature, buffer, and pH for E. coli K-12 AHAS I were 37 °C, potassium phosphate buffer, and 7.5. Km values for E. coli K-12 AHAS I binding to pyruvate, Mg(+2), ThDP, and FAD were 4.15, 1.26, 0.2 mM, and 0.61 µM respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.

  16. Proteins involved in wine aroma compounds metabolism by a Saccharomyces cerevisiae flor-velum yeast strain grown in two conditions.

    PubMed

    Moreno-García, Jaime; García-Martínez, Teresa; Millán, M Carmen; Mauricio, Juan Carlos; Moreno, Juan

    2015-10-01

    A proteomic and exometabolomic study was conducted on Saccharomyces cerevisiae flor yeast strain growing under biofilm formation condition (BFC) with ethanol and glycerol as carbon sources and results were compared with those obtained under no biofilm formation condition (NBFC) containing glucose as carbon source. By using modern techniques, OFFGEL fractionator and LTQ-Orbitrap for proteome and SBSE-TD-GC-MS for metabolite analysis, we quantified 84 proteins including 33 directly involved in the metabolism of glycerol, ethanol and 17 aroma compounds. Contents in acetaldehyde, acetic acid, decanoic acid, 1,1-diethoxyethane, benzaldehyde and 2-phenethyl acetate, changed above their odor thresholds under BFC, and those of decanoic acid, ethyl octanoate, ethyl decanoate and isoamyl acetate under NBFC. Of the twenty proteins involved in the metabolism of ethanol, acetaldehyde, acetoin, 2,3-butanediol, 1,1-diethoxyethane, benzaldehyde, organic acids and ethyl esters, only Adh2p, Ald4p, Cys4p, Fas3p, Met2p and Plb1p were detected under BFC and as many Acs2p, Ald3p, Cem1p, Ilv2p, Ilv6p and Pox1p, only under NBFC. Of the eight proteins involved in glycerol metabolism, Gut2p was detected only under BFC while Pgs1p and Rhr2p were under NBFC. Finally, of the five proteins involved in the metabolism of higher alcohols, Thi3p was present under BFC, and Aro8p and Bat2p were under NBFC. PMID:26187821

  17. High genetic diversity among strains of the unindustrialized lactic acid bacterium Carnobacterium maltaromaticum in dairy products as revealed by multilocus sequence typing.

    PubMed

    Rahman, Abdur; Cailliez-Grimal, Catherine; Bontemps, Cyril; Payot, Sophie; Chaillou, Stéphane; Revol-Junelles, Anne-Marie; Borges, Frédéric

    2014-07-01

    Dairy products are colonized with three main classes of lactic acid bacteria (LAB): opportunistic bacteria, traditional starters, and industrial starters. Most of the population structure studies were previously performed with LAB species belonging to these three classes and give interesting knowledge about the population structure of LAB at the stage where they are already industrialized. However, these studies give little information about the population structure of LAB prior their use as an industrial starter. Carnobacterium maltaromaticum is a LAB colonizing diverse environments, including dairy products. Since this bacterium was discovered relatively recently, it is not yet commercialized as an industrial starter, which makes C. maltaromaticum an interesting model for the study of unindustrialized LAB population structure in dairy products. A multilocus sequence typing scheme based on an analysis of fragments of the genes dapE, ddlA, glpQ, ilvE, pyc, pyrE, and leuS was applied to a collection of 47 strains, including 28 strains isolated from dairy products. The scheme allowed detecting 36 sequence types with a discriminatory index of 0.98. The whole population was clustered in four deeply branched lineages, in which the dairy strains were spread. Moreover, the dairy strains could exhibit a high diversity within these lineages, leading to an overall dairy population with a diversity level as high as that of the nondairy population. These results are in agreement with the hypothesis according to which the industrialization of LAB leads to a diversity reduction in dairy products.

  18. Frequencies of mutagen-induced coincident mitotic recombination at unlinked loci in Saccharomyces cerevisiae.

    PubMed

    Freeman, Kathryn M; Hoffmann, George R

    2007-03-01

    Frequencies of coincident genetic events were measured in strain D7 of Saccharomyces cerevisiae. This diploid strain permits the detection of mitotic gene conversion involving the trp5-12 and trp5-27 alleles, mitotic crossing-over and gene conversion leading to the expression of the ade2-40 and ade2-119 alleles as red and pink colonies, and reversion of the ilv1-92 allele. The three genes are on different chromosomes, and one might expect that coincident (simultaneous) genetic alterations at two loci would occur at frequencies predicted by those of the single alterations acting as independent events. Contrary to this expectation, we observed that ade2 recombinants induced by bleomycin, beta-propiolactone, and ultraviolet radiation occur more frequently among trp5 convertants than among total colonies. This excess among trp5 recombinants indicates that double recombinants are more common than expected for independent events. No similar enrichment was found among Ilv(+) revertants. The possibility of an artifact in which haploid yeasts that mimic mitotic recombinants are generated by a low frequency of cryptic meiosis has been excluded. Several hypotheses that can explain the elevated incidence of coincident mitotic recombination have been evaluated, but the cause remains uncertain. Most evidence suggests that the excess is ascribable to a subset of the population being in a recombination-prone state. PMID:17156798

  19. On multi-timescale variability of temperature in China in modulated annual cycle reference frame

    NASA Astrophysics Data System (ADS)

    Qian, Cheng; Wu, Zhaohua; Fu, Congbin; Zhou, Tianjun

    2010-09-01

    The traditional anomaly (TA) reference frame and its corresponding anomaly for a given data span changes with the extension of data length. In this study, the modulated annual cycle (MAC), instead of the widely used climatological mean annual cycle, is used as an alternative reference frame for computing climate anomalies to study the multi-timescale variability of surface air temperature (SAT) in China based on homogenized daily data from 1952 to 2004. The Ensemble Empirical Mode Decomposition (EEMD) method is used to separate daily SAT into a high frequency component, a MAC component, an interannual component, and a decadal-to-trend component. The results show that the EEMD method can reflect historical events reasonably well, indicating its adaptive and temporally local characteristics. It is shown that MAC is a temporally local reference frame and will not be altered over a particular time span by an extension of data length, thereby making it easier for physical interpretation. In the MAC reference frame, the low frequency component is found more suitable for studying the interannual to longer timescale variability (ILV) than a 13-month window running mean, which does not exclude the annual cycle. It is also better than other traditional versions (annual or summer or winter mean) of ILV, which contains a portion of the annual cycle. The analysis reveals that the variability of the annual cycle could be as large as the magnitude of interannual variability. The possible physical causes of different timescale variability of SAT in China are further discussed.

  20. Crystal structures of RidA, an important enzyme for the prevention of toxic side products

    PubMed Central

    Liu, Xiwen; Zeng, Jianhua; Chen, Xiaolei; Xie, Wei

    2016-01-01

    The YjgF/YER057c/UK114 family proteins are highly conserved across all three domains of life, and most of them currently have no clearly defined biological roles. In vitro, these proteins were found to hydrolyze the enamine/imine intermediates generated from serine or threonine, and were renamed Reactive Intermediate Deaminase A (RidA). RidA was recently discovered in Arabidopsis thaliana, and by deaminating the toxic enamine/imine intermediates, it prevents the inactivation of many functionally important pyridoxal 5′-phosphate (PLP)-containing enzymes in plants such as branched-chain aminotransferase BCAT (IlvE). In this study, we determined the crystal structure of Arabidopsis thaliana RidA in the apo form, as well as RidA complexed with the ligand pyruvate. RidA forms the trimeric, barrel-like quaternary structure and inter-subunit cavities, and resembles most RidA family members. Each pyruvate molecule binds to the interface between two subunits, and the recognition of pyruvate is achieved by the interactions with R165 and T167. From sequence alignment and structural superposition, we identified a series of key residues responsible for the trimer assembly, whose importance was confirmed by enzymatic assays. This study provides structural insight into RidA functions in plants. PMID:27458092

  1. Antimutagenic properties of green tea.

    PubMed

    Bunkova, R; Marova, I; Nemec, M

    2005-03-01

    In this work biological effects of two common kinds of green tea (Chinese Gunpowder and Japanese Sencha) were analyzed using three independent tests of antimutagenicity: 1) the Ames test with Salmonella typhimurium TA98, 2) cytogenetic analysis of peripheral blood lymphocytes (CAPL), and 3) test with Saccharomyces cerevisiae D7. Tea extracts were allowed to be antimutagenic based on their ability to inhibit the mutagenic effect of standard mutagens. Amounts of (-)catechin and (-)catechin gallate in tea extracts were determined by high performance liquid chromatography on reversed phase (RP-HPLC). Antioxidant capacity was found using total radical-trapping antioxidant parameter (TRAP) method. Extracts from Gunpowder and Sencha exhibited high antimutagenic activity in the Ames test (24.7+/-3.7% and 34.1+/-2, 1% of inhibition without metabolic activation; 74.9+/-1.7% and 62.7+/-4.3% of inhibition with metabolic activation, respectively) as well as in S. cerevisiae D7 test (Gunpowder: 62.7+/-5.7% of Trp convertants inhibition and 52.6+/-5.3% of Ilv revertants inhibition; Sencha: 45.6+/-4.2% of Trp convertants inhibition, 50.0+/-4.8% of Ilv revertants inhibition). In the CAPL method reduced number of abberant cells as well as decreased number of chromosome breaks was observed using both green tea extracts. Antioxidant capacity and antimutagenicity of green tea extracts was higher than activity of tea catechins and flavonoids.

  2. Characterization of hybrid plasmids carrying individual ribosomal ribonucleic acid transcription units of Escherichia coli.

    PubMed Central

    Kenerley, M E; Morgan, E A; Post, L; Lindahl, L; Nomura, M

    1977-01-01

    We have screened the strains with ColE1 hybrid plasmids constructed by Clarke and Carbon (Cell 9:91-99, 1976) for the presence of ribosomal ribonucleic acid (rRNA) genes on the plasmids and identified 16 strains whose plasmids carry rRNA genes. The structures of these 16 plasmids were compared by heteroduplex analysis, and the plasmids were classified into six groups on the basis of their chromosomal origins. Homology with known transducing-phage deoxyribonucleic acids and genetic mapping have assigned locations on the Escherichia coli chromosome to three of the six groups. These are rrnB near rif at 88 min, rrnC near ilvE at 83 min, and rrnD near aroE at 71 min. A fourth group is probably rrnA at 85 min (T. Ikemura and M. Nomura, Cell, 11:779-793, 1977). We conclude that the minimum number of rRNA transcription units per haploid chromosomes is seven, that is, the six groups identified in this work plus a known operon (rrnE near metA at 89 min) that we failed to find among the hybrid plasmids. This heteroduplex analysis also suggests that there are only two kinds of rRNA operons with respect to their spacer region; three of the six rRNA operon groups studied here have one kind, whereas the remaining three have the other kind. Images PMID:336613

  3. System identification of closed-loop cardiovascular control mechanisms: diabetic autonomic neuropathy

    NASA Technical Reports Server (NTRS)

    Mukkamala, R.; Mathias, J. M.; Mullen, T. J.; Cohen, R. J.; Freeman, R.

    1999-01-01

    We applied cardiovascular system identification (CSI) to characterize closed-loop cardiovascular regulation in patients with diabetic autonomic neuropathy (DAN). The CSI method quantitatively analyzes beat-to-beat fluctuations in noninvasively measured heart rate, arterial blood pressure (ABP), and instantaneous lung volume (ILV) to characterize four physiological coupling mechanisms, two of which are autonomically mediated (the heart rate baroreflex and the coupling of respiration, measured in terms of ILV, to heart rate) and two of which are mechanically mediated (the coupling of ventricular contraction to the generation of the ABP wavelet and the coupling of respiration to ABP). We studied 37 control and 60 diabetic subjects who were classified as having minimal, moderate, or severe DAN on the basis of standard autonomic tests. The autonomically mediated couplings progressively decreased with increasing severity of DAN, whereas the mechanically mediated couplings were essentially unchanged. CSI identified differences between the minimal DAN and control groups, which were indistinguishable based on the standard autonomic tests. CSI may provide a powerful tool for assessing DAN.

  4. Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes.

    PubMed

    Mazzeo, C; Calvo, V; Alonso, R; Mérida, I; Izquierdo, M

    2016-01-01

    Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as 'lethal exosomes' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic.

  5. Genetic Analysis of Cold-Sensitive Ribosome Maturation Mutants of Escherichia coli

    PubMed Central

    Bryant, Robert E.; Sypherd, Paul S.

    1974-01-01

    Four cold-sensitive mutants of Escherichia coli, which have defects in the maturation of the 50S ribosomal subunit, were isolated. Each of the mutations was shown to map at a different locus. The loci were assigned the name rim (ribosome maturation) and were shown to map as follows: rimA is co-transduced with ilvD and with pyrE; rimB is co-transduced with aroD; conjugation experiments limited rimD to a region between ilv and malB, and conjugation experiments limited rimC to the 22 to 30 min region of the chromosome. In merodiploids heterozygous for rimA, rimB, or rimD, the wild-type allele was shown to be dominant to the mutant allele. The observation that the rim loci lie outside the strA region and separate from each other, as well as the recessive character of the rim loci, suggests that the mutants may be defective in ribosome maturation factors rather than being defective in ribosomal structural proteins. PMID:4591943

  6. Systematic investigations of the stable Cd isotopes

    NASA Astrophysics Data System (ADS)

    Garrett, P. E.; Green, K. L.; Wood, J. L.; Kulp, W. D.

    2007-10-01

    The Cd nuclei, especially the stable even-even isotopes have been well studied since they were suggested as paradigms of the vibrational, or U(5), limit of the Interacting Boson Model (IBM). In addition to the normal quadrupole phonon states, in many cases suggested up to the three-phonon quintuplet, more deformed 2p4h intruder excitations have been established. Recent investigations with the (n,n^'γ) reaction [1,2,3,4] have provided a wealth of information on the low-lying levels, including many lifetimes not previously known. Deviations in the transition B(E2) values for low-spin states from those expected for U(5) nuclei are observed to appear systematically across the Cd isotopes. We have performed detailed calculations using the IBM-2, and find that these deviations cannot be explained through considered mixings with the intruder excitations or mixed-symmetry states, indicating that some physics is missing in the description of these levels. [1] F. Corminboeuf et al., Phys. Rev. C 63, 014305 (2001).[2] P.E. Garrett et al., Phys. Rev. C 75, 014307 (2007).[3] D. Bandyopadhyay et al., to be published. [4] M. Kadi et al., Phys. Rev. 68, 031306 (2003).

  7. Food incentives for sterilization: can they be just?

    PubMed

    Pohlman, E; Callahan, D

    1973-02-01

    The Indian Family Planning Council has recommended an incentive of 100 kadis worth of food to anyone undergoing sterilization as a means of limiting population growth. Proponents of this incentive system acknowledge that such a system may be considered a limitation of freedom but they propose that greater restrictions of freedom are the result of excessive populations because of limited health care, poor nutrition, and few educational opportunities. Since society manipulates other aspects of private life, it has the right do try to limit family size since high rates of population growth are harmful to the society as a whole. Opposition to the incentive scheme is based on the idea of a permanent benefit for a temporary bonus. Also it is argued that since the poor are the ones to be chiefly affected by incentives, their decision is a forced choice because they must decide between no food or no children. Only if it could be proved that the population problem is the most important problem facing the nation, could such incentive plans perhaps be justified.

  8. Recharge Data for the Islands of Kauai, Lanai and Molokai, Hawaii

    DOE Data Explorer

    Nicole Lautze

    2015-01-01

    Recharge data for the islands of Kauai, Lanai and Molokai in shapefile format. These data are from the following sources: Whittier, R.B and A.I. El-Kadi. 2014. Human Health and Environmental Risk Ranking of On-Site Sewage Disposal systems for the Hawaiian Islands of Kauai, Molokai, Maui, and Hawaii – Final, Prepared for Hawaii Dept. of Health, Safe Drinking Water Branch by the University of Hawaii, Dept. of Geology and Geophysics. (for Kauai, Lanai, Molokai). Shade, P.J., 1995, Water Budget for the Island of Kauai, Hawaii, USGS Water-Resources Investigations Report 95-4128, 25 p. (for Kauai). Izuka, S.K. and D.S. Oki, 2002 Numerical simulation of ground-water withdrawals in the Southern Lihue Basin, Kauai, Hawaii, U.S. Geologic Survey Water-Resources Investigations Report 01-4200, 52 pgs. (for Kauai). Hardy, W.R., 1996, A Numerical Groundwater Model for the Island of Lanai, Hawaii - CWRM Report No., CWRM-1, Commission on Water Resources Management, Department of Natural Resources, State of Hawaii, Honolulu, HI. (for Lanai). Oki, D.S., 1997, Geohydrology and numerical Simulation of the Ground-Water Flow System of Molokai, Hawaii, USGS Water-Resources Investigations Report 97-4176, 62 p. (for Molokai).

  9. Role of the host cell in bacteriophage T4 development. II. Characterization of host mutants that have pleiotropic effects on T4 growth.

    PubMed Central

    Stitt, B L; Revel, H R; Lielausis, I; Wood, W B

    1980-01-01

    Mutant host-defective Escherichi coli that fail to propagate bacteriophage T4 and have a pleiotropic effect on T4 development have been isolated and characterized. In phage-infected mutant cells, specific early phage proteins are absent or reduced in amount, phage DNA synthesis is depressed by about 50%, specific structural phage proteins, including some tail and collar components, are deficient or missing, and host-cell lysis is delayed and slow. Almost all phage that can overcome the host block carry mutantions that map in functionally undefined 'nonessential' regions of the T4 genome, most near gene 39. The mutant host strains are temperature sensitive for growth and show simultaneous reversion of the ts phenotype and the inability to propagate T4+. The host mutations are cotransduced with ilv (83 min) and may lie in the gene for transcription termination factor rho. Images PMID:6999171

  10. Diacetyl and acetoin production from whey permeate using engineered Lactobacillus casei.

    PubMed

    Nadal, Inmaculada; Rico, Juan; Pérez-Martínez, Gaspar; Yebra, María J; Monedero, Vicente

    2009-09-01

    The capability of Lactobacillus casei to produce the flavor-related compounds diacetyl and acetoin from whey permeate has been examined by a metabolic engineering approach. An L. casei strain in which the ilvBN genes from Lactococcus lactis, encoding acetohydroxyacid synthase, were expressed from the lactose operon was mutated in the lactate dehydrogenase gene (ldh) and in the pdhC gene, which codes for the E2 subunit of the pyruvate dehydrogenase complex. The introduction of these mutations resulted in an increased capacity to synthesize diacetyl/acetoin from lactose in whey permeate (1,400 mg/l at pH 5.5). The results showed that L. casei can be manipulated to synthesize added-value metabolites from dairy industry by-products.

  11. Diacetyl and acetoin production from whey permeate using engineered Lactobacillus casei.

    PubMed

    Nadal, Inmaculada; Rico, Juan; Pérez-Martínez, Gaspar; Yebra, María J; Monedero, Vicente

    2009-09-01

    The capability of Lactobacillus casei to produce the flavor-related compounds diacetyl and acetoin from whey permeate has been examined by a metabolic engineering approach. An L. casei strain in which the ilvBN genes from Lactococcus lactis, encoding acetohydroxyacid synthase, were expressed from the lactose operon was mutated in the lactate dehydrogenase gene (ldh) and in the pdhC gene, which codes for the E2 subunit of the pyruvate dehydrogenase complex. The introduction of these mutations resulted in an increased capacity to synthesize diacetyl/acetoin from lactose in whey permeate (1,400 mg/l at pH 5.5). The results showed that L. casei can be manipulated to synthesize added-value metabolites from dairy industry by-products. PMID:19609583

  12. Characterization and mutagenesis of two novel iron-sulphur cluster pentonate dehydratases.

    PubMed

    Andberg, Martina; Aro-Kärkkäinen, Niina; Carlson, Paul; Oja, Merja; Bozonnet, Sophie; Toivari, Mervi; Hakulinen, Nina; O'Donohue, Michael; Penttilä, Merja; Koivula, Anu

    2016-09-01

    We describe here the identification and characterization of two novel enzymes belonging to the IlvD/EDD protein family, the D-xylonate dehydratase from Caulobacter crescentus, Cc XyDHT, (EC 4.2.1.82), and the L-arabonate dehydratase from Rhizobium leguminosarum bv. trifolii, Rl ArDHT (EC 4.2.1.25), that produce the corresponding 2-keto-3-deoxy-sugar acids. There is only a very limited amount of characterization data available on pentonate dehydratases, even though the enzymes from these oxidative pathways have potential applications with plant biomass pentose sugars. The two bacterial enzymes share 41 % amino acid sequence identity and were expressed and purified from Escherichia coli as homotetrameric proteins. Both dehydratases were shown to accept pentonate and hexonate sugar acids as their substrates and require Mg(2+) for their activity. Cc XyDHT displayed the highest activity on D-xylonate and D-gluconate, while Rl ArDHT functioned best on D-fuconate, L-arabonate and D-galactonate. The configuration of the OH groups at C2 and C3 position of the sugar acid were shown to be critical, and the C4 configuration also contributed substantially to the substrate recognition. The two enzymes were also shown to contain an iron-sulphur [Fe-S] cluster. Our phylogenetic analysis and mutagenesis studies demonstrated that the three conserved cysteine residues in the aldonic acid dehydratase group of IlvD/EDD family members, those of C60, C128 and C201 in Cc XyDHT, and of C59, C127 and C200 in Rl ArDHT, are needed for coordination of the [Fe-S] cluster. The iron-sulphur cluster was shown to be crucial for the catalytic activity (kcat) but not for the substrate binding (Km) of the two pentonate dehydratases. PMID:27102126

  13. Clusters of branched aliphatic side chains serve as cores of stability in the native state of the HisF TIM barrel protein.

    PubMed

    Gangadhara, Basavanapura N; Laine, Jennifer M; Kathuria, Sagar V; Massi, Francesca; Matthews, C Robert

    2013-03-25

    Imidazole-3-glycerol phosphate synthase is a heterodimeric allosteric enzyme that catalyzes consecutive reactions in imidazole biosynthesis through its HisF and HisH subunits. The unusually slow unfolding reaction of the isolated HisF TIM barrel domain from the thermophilic bacteria, Thermotoga maritima, enabled an NMR-based site-specific analysis of the main-chain hydrogen bonds that stabilize its native conformation. Very strong protection against exchange with solvent deuterium in the native state was found in a subset of buried positions in α-helices and pervasively in the underlying β-strands associated with a pair of large clusters of isoleucine, leucine and valine (ILV) side chains located in the α7(βα)8(βα)1-2 and α2(βα)3-6β7 segments of the (βα)8 barrel. The most densely packed region of the large cluster, α3(βα)4-6β7, correlates closely with the core of stability previously observed in computational, protein engineering and NMR dynamics studies, demonstrating a key role for this cluster in determining the thermodynamic and structural properties of the native state of HisF. When considered with the results of previous studies where ILV clusters were found to stabilize the hydrogen-bonded networks in folding intermediates for other TIM barrel proteins, it appears that clusters of branched aliphatic side chains can serve as cores of stability across the entire folding reaction coordinate of one of the most common motifs in biology.

  14. Characterization of genetically transformed Saccharomyces cerevisiae baker's yeasts able to metabolize melibiose.

    PubMed Central

    Gasent-Ramírez, J M; Codón, A C; Benítez, T

    1995-01-01

    Three transformant (Mel+) Saccharomyces cerevisiae baker's yeast strains, CT-Mel, VS-Mel, and DADI-Mel, have been characterized. The strains, which originally lacked alpha-galactosidase activity (Mel-), had been transformed with a DNA fragment which possessed an ILV1-SMR1 allele of the ILV2 gene and a MEL1 gene. The three transformed strains showed growth rates similar to those of the untransformed controls in both minimal and semi-industrial (molasses) media. The alpha-galactosidase specific activity of strain CT-Mel was twice that of VS-Mel and DADI-Mel. The yield, YX/S (milligrams of protein per milligram of substrate), in minimal medium with raffinose as the carbon source was 2.5 times higher in the transformed strains than in the controls and was 1.5 times higher in CT-Mel than in VS-Mel and DADI-Mel. When molasses was used, YX/S (milligrams of protein per milliliter of culture) increased 8% when the transformed strains CT-Mel and DADI-Mel were used instead of the controls. Whereas no viable spores were recovered from either DADI-Mel or VS-Mel tetrads, genetic analysis carried out with CT-Mel indicated that the MEL1 gene has been integrated in two of three homologous loci. Analysis of the DNA content by flow cytometry indicated that strain CT-Mel was 3n, whereas VS-Mel was 2n and DADI-Mel was 1.5n. Electrophoretic karyotype and Southern blot analyses of the transformed strains showed that the MEL1 gene has been integrated in the same chromosomic band, probably chromosome XIII, in the three strains.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7793932

  15. Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

    SciTech Connect

    Lu, JN; Brigham, CJ; Gai, CS; Sinskey, AJ

    2012-08-04

    Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.

  16. A consensus sequence for binding of Lrp to DNA.

    PubMed Central

    Cui, Y; Wang, Q; Stormo, G D; Calvo, J M

    1995-01-01

    Lrp (leucine-responsive regulatory protein) is a major regulatory protein involved in the expression of numerous operons in Escherichia coli. For ilvIH, one of the operons positively regulated by Lrp, Lrp binds to multiple sites upstream of the transcriptional start site and activates transcription. An alignment of 12 Lrp binding sites within ilvIH DNA from two different organisms revealed a tentative consensus sequence AGAAT TTTATTCT (Q. Wang, M. Sacco, E. Ricca, C.T. Lago, M. DeFelice, and J.M. Calvo, Mol. Microbiol. 7:883-891, 1993). To further characterize the binding specificity of Lrp, we used a variation of the Selex procedure of C. Tuerk and L. Gold (Science 249:505-510, 1990) to identify sequences that bound Lrp out of a pool of 10(12) different DNA molecules. We identified 63 related DNA sequences that bound Lrp and estimated their relative binding affinities for Lrp. A consensus sequence derived from analysis of these sequences, YAGHAWATTWT DCTR, where Y = C or T, H = not G, W = A or T, D = not C, and R = A or G, contains clear dyad symmetry and is very similar to the one defined earlier. To test the idea that Lrp in the presence of leucine might bind to a different subset of DNA sequences, we carried out a second selection experiment with leucine present during the binding reactions. DNA sequences selected in the presence or absence of leucine were similar, and leucine did not stimulate binding to any of the sequences that were selected in the presence of leucine. Therefore, it is unlikely that leucine changes the specificity of Lrp binding. PMID:7665463

  17. [Development of internal medicine since the establishment of the Regional Hospital in Sarajevo from 1894 until today].

    PubMed

    Grujić, M

    1994-01-01

    The first bigger regular hospital in Sarajevo was built by the Bosnian vali Osman-pasha in the year 1866 and had only 32 beds. The construction expenses and keeping, according the Porta's approval was supported by Gazi-Husrevbeg's Vakuf, the name was The Vakuf's Hospital. In the year 1894, initiated by the Regional Government (Landesregierung) the Regional Hospital (Landesspital) came to existence, 1894, when opened had 238 beds, with modern outfits and technical facilities. There were 4 departments: Internal, Surgery, Dermatovenerology and Gynecology-Obstetrics. The Department for Internal Diseases had 50 beds. The first chief was Primarius Dr Geza Kobler, came from the Viennese Medical Faculty and had been assistant to Prof. Schretter. Simultaneously, he was director of the Hospital. His assistants were: Dr Berthold Haas to 1898 and Dr Heinrich Propper to 1900 and the interns were: Dr Bodo Koloman, Dr Alexander Dorner, Dr Erwin Horn, Dr Dragan Plentaj and Dr Risto Jeremić. Later, as chief was appointed Dr Ludomil Sas-Korcinski, professor of the Cracau University, his associates were to the year 1910: Dr Hamdija Karamehmedović, later to became chief of the Department for Infectious Diseases and the Intern, Dr Mustafa Kadić. At the eve of the World War 1, in 1914, the pavilion "K" was built, where the Department housed. At that time chief was Primarius Dr Ljubo Bilić, while his associates were Dr Gavro Bozić, Dr Vjekoslav Kusan and Dr Duro Ostojić. Later, some other doctors same, having a permanent job, Dr Bogdan Zimonjić, Dr Miron Simić, Dr David Pinto, Dr Sreten Kaluräercić and Dr David Baruh.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Activation of satellite cells and the regeneration of human skeletal muscle are expedited by ingestion of nonsteroidal anti-inflammatory medication.

    PubMed

    Mackey, Abigail L; Rasmussen, Lotte K; Kadi, Fawzi; Schjerling, Peter; Helmark, Ida C; Ponsot, Elodie; Aagaard, Per; Durigan, João Luiz Q; Kjaer, Michael

    2016-06-01

    With this study we investigated the role of nonsteroidal anti-inflammatory drugs (NSAIDs) in human skeletal muscle regeneration. Young men ingested NSAID [1200 mg/d ibuprofen (IBU)] or placebo (PLA) daily for 2 wk before and 4 wk after an electrical stimulation-induced injury to the leg extensor muscles of one leg. Muscle biopsies were collected from the vastus lateralis muscles before and after stimulation (2.5 h and 2, 7, and 30 d) and were assessed for satellite cells and regeneration by immunohistochemistry and real-time RT-PCR, and we also measured telomere length. After injury, and compared with PLA, IBU was found to augment the proportion of ActiveNotch1(+) satellite cells at 2 d [IBU, 29 ± 3% vs. PLA, 19 ± 2% (means ± sem)], satellite cell content at 7 d [IBU, 0.16 ± 0.01 vs. PLA, 0.12 ± 0.01 (Pax7(+) cells/fiber)], and to expedite muscle repair at 30 d. The PLA group displayed a greater proportion of embryonic myosin(+) fibers and a residual ∼2-fold increase in mRNA levels of matrix proteins (all P < 0.05). Endomysial collagen was also elevated with PLA at 30 d. Minimum telomere length shortening was not observed. In conclusion, ingestion of NSAID has a potentiating effect on Notch activation of satellite cells and muscle remodeling during large-scale regeneration of injured human skeletal muscle.-Mackey, A. L., Rasmussen, L. K., Kadi, F., Schjerling, P., Helmark, I. C., Ponsot, E., Aagaard, P., Durigan, J. L. Q., Kjaer, M. Activation of satellite cells and the regeneration of human skeletal muscle are expedited by ingestion of nonsteroidal anti-inflammatory medication. PMID:26936358

  19. Effective Trapping of Fruit Flies with Cultures of Metabolically Modified Acetic Acid Bacteria

    PubMed Central

    Ishii, Yuri; Akasaka, Naoki; Goda, Itsuko; Sakoda, Hisao

    2015-01-01

    Acetoin in vinegar is an attractant to fruit flies when combined with acetic acid. To make vinegar more effective in attracting fruit flies with increased acetoin production, Komagataeibacter europaeus KGMA0119 was modified by specific gene disruption of the acetohydroxyacid isomeroreductase gene (ilvC). A previously constructed mutant lacking the putative ligand-sensing region in the leucine-responsive regulatory protein (KeLrp, encoded by Kelrp) was also used. The ilvC and Kelrp disruptants (KGMA5511 and KGMA7203, respectively) produced greater amounts of acetoin (KGMA5511, 0.11%; KGMA7203, 0.13%) than the wild-type strain KGMA0119 (0.069%). KGMA7203 produced a trace amount of isobutyric acid (0.007%), but the other strains did not. These strains produced approximately equal amounts of acetic acid (0.7%). The efficiency of fruit fly attraction was investigated with cultured Drosophila melanogaster. D. melanogaster flies (approximately 1,500) were released inside a cage (2.5 m by 2.5 m by 1.5 m) and were trapped with a device containing vinegar and a sticky sheet. The flies trapped on the sticky sheet were counted. The cell-free supernatant from KGMA7203 culture captured significantly more flies (19.36 to 36.96% of released flies) than did KGMA0119 (3.25 to 11.40%) and KGMA5511 (6.87 to 21.50%) cultures. Contrastingly, a 0.7% acetic acid solution containing acetoin (0.13%) and isobutyric acid (0.007%), which mimicked the KGMA7203 supernatant, captured significantly fewer flies (0.88 to 4.57%). Furthermore, the KGMA0119 supernatant with additional acetoin (0.13%) and isobutyric acid (0.007%) captured slightly more flies than the original KGMA0119 supernatant but fewer than the KGMA7203 supernatant, suggesting that the synergistic effects of acetic acid, acetoin, isobutyric acid, and unidentified metabolites achieved the efficient fly trapping of the KGMA7203 supernatant. PMID:25595769

  20. Viral miRNAs exploiting the endosomal-exosomal pathway for intercellular cross-talk and immune evasion.

    PubMed

    Pegtel, D Michiel; van de Garde, Martijn D B; Middeldorp, Jaap M

    2011-01-01

    The class of persistent gamma-herpesviruses has developed a variety of strategies that exploit host-cell regulatory pathways to ensure a long-lasting, well-balanced infection of their host. However when these pathways are deregulated, an otherwise harmless infection can lead to disease including cancer. We recently demonstrated that the human herpes virus 4 (HHV4) also known as Epstein-Barr virus (EBV), encodes for small regulatory non-coding microRNAs (miRNAs) that can be transferred from an infected cell to uninfected neighboring cells. Upon arrival these miRNAs are functional in the recipient cell, in that they are able to down regulate specific target genes. These secreted miRNAs are transported to recipient cells via small nano-sized vesicles (known as exosomes) that are of endosomal origin, formed as intraluminal vesicles (ILV) inside multivesicular bodies (MVB). One question that needs to be addressed is how viral miRNAs are sorted into these exosomes. Mature miRNAs, including those of viral origin, are loaded into RNA-induced silencing complexes (RISC) for gene silencing via blocking mRNA translation and/or initiating mRNA decay. Recent insights indicate that cytoplasmic RNA granules rich in RISC complexes are closely associated with endosomes. In fact, selective components of RISC, including GW182 and Argonaut proteins, miRNAs and mRNAs are present in exosomes. Thus miRNA function, mRNA stability and exosome-mediated intercellular communication converge at the level of endosomes. Since endosomes can be considered as key intracellular cross-roads that regulate communication of cells with their exterior, including neighboring cells, it is perhaps not surprising that viruses have found means to exploit this pathway to their benefit. Little is known however, how and if (micro) RNA species are specifically sorted into ILVs and what (micro)RNA-binding proteins are involved. Here we discuss recent developments relating to intracellular trafficking and function of

  1. Estimate of Gaseous 14Carbon Concentrations Emanating from the Intermediate-Level Vault Disposal Facility

    SciTech Connect

    Kaplan, D

    2005-08-31

    {sup 14}Carbon-bearing resin waste will be disposed in the Low-Activity Waste (LAW) Intermediate Level Vaults (ILV) located in E-Area on the Savannah River Site (SRS). This waste will be buried in a cementitious environment in the vadose zone, i.e., the subsurface zone above the aquifer. As the resin ages, and equilibrates with slowly infiltrating water, it is expected that the {sup 14}C will partition to the solid, liquid, and gaseous phases. The objective of this task was to estimate the concentration of gaseous {sup 14}C in the waste pore space that is in contact with the resin leachate. The approach used to estimate this value was built largely around data generated from lysimeter studies that were conducted for 9 years. These lysimeters contained the same type of used resins (mixed-bed deionizer resins used in the purification of the heavy water moderator of SRS reactors) as are being disposed in the ILV. During the 9 year period, pore water {sup 14}C leaching concentrations were monitored to provide an excellent estimate of the long-term behavior of {sup 14}C release rates from the resins. Thermodynamic calculations were conducted to calculate {sup 14}CO{sub 2(g)} concentrations. These calculations included the {sup 14}C pore water data from the lysimeter study, and data from a field study that was a natural analogue to a long-term cementitious environment (Khoury et al. 1992). The calculations predicted an extremely low {sup 14}CO{sub 2(g)} concentration of 1.9 x 10{sup -7} Ci/m{sup 3} {sup 14}CO{sub 2(g)} in the air spaces above the resin leachate. This low concentrations is not surprising in light of both laboratory and field observations that concrete acts as a strong sorbent of CO{sub 2(g)}. This calculated {sup 14}CO{sub 2(g)} concentration will now be included in future risk calculations.

  2. Avoiding the Water-Climate-Poverty Trap: Adaptive Risk Management for Bangladesh's Coastal Embankments

    NASA Astrophysics Data System (ADS)

    Hall, J. W.

    2015-12-01

    ., Aerts, J.C.J.H., Ait-Kadi, M., Brown, C., Cox, A., Dadson, S., Garrick, D., Kelman, J., McCornick, P., Ringler, C., Rosegrant, M., Whittington, D. and Wiberg, D. Securing Water, Sustaining Growth: Report of the GWP/OECD Task Force on Water Security and Sustainable Growth, University of Oxford, April 2015, 180pp.

  3. Metabolic engineering of Escherichia coli for the production of 1-propanol.

    PubMed

    Choi, Yong Jun; Park, Jin Hwan; Kim, Tae Yong; Lee, Sang Yup

    2012-09-01

    An engineered Escherichia coli strain that produces 1-propanol under aerobic condition was developed based on an L-threonine-overproducing E. coli strain. First, a feedback resistant ilvA gene encoding threonine dehydratase was introduced and the competing metabolic pathway genes were deleted. Further engineering was performed by overexpressing the cimA gene encoding citramalate synthase and the ackA gene encoding acetate kinase A/propionate kinase II, introducing a modified adhE gene encoding an aerobically functional AdhE, and by deleting the rpoS gene encoding the stationary phase sigma factor. Fed-batch culture of the final engineered strain harboring pBRthrABC-tac-cimA-tac-ackA and pTacDA-tac-adhE(mut) allowed production of 10.8 g L(-1) of 1-propanol with the yield and productivity of 0.107 g g(-1) and 0.144 g L(-1) h(-1), respectively, from 100 g L(-1) of glucose, and 10.3 g L(-1) of 1-propanol with the yield and productivity of 0.259 g g(-1) and 0.083 g L(-1) h(-1), respectively, from 40 g L(-1) glycerol. PMID:22871504

  4. Multilocus sequence typing of Campylobacter concisus from Danish diarrheic patients.

    PubMed

    Nielsen, Hans Linde; Nielsen, Henrik; Torpdahl, Mia

    2016-01-01

    The emerging enteric pathogen Campylobacter concisus is associated with prolonged diarrhea and inflammatory bowel disease. Previous studies have shown that C. concisus strains are very genetically diverse. Nevertheless, C. concisus strains have been divided into two genomospecies, where GS1 strains have been isolated predominantly from healthy individuals, while the GS2 cluster consists of isolates primarily from diarrheic individuals. The aim of the present study was to determine the genetic diversity of C. concisus isolates from Danish diarrheic patients. Multilocus sequence typing using the loci aspA, atpA, glnA, gltA, glyA, ilvD and pgm, as well as genomospecies based on specific differences in the 23S rRNA, was used to characterize 67 isolates (63 fecal and 4 oral), from 49 patients with different clinical presentations (29 with diarrhea, eight with bloody diarrhea, seven with collagenous colitis and five with Crohn's disease). MLST revealed a high diversity of C. concisus with 53 sequence types (STs), of which 52 were identified as 'new' STs. Allele sequences showed more than 90 % similarity between isolates, with only four outliers. Dendrogram profiles of each allele showed a division into two groups, which more or less correlated with genomospecies A and genomospecies B. However, in contrary to previous results, this subgrouping had no association to the clinical severity of disease. PMID:27688814

  5. Characterization of D-xylonate dehydratase YjhG from Escherichia coli

    PubMed Central

    Jiang, Yudong; Liu, Wei; Cheng, Tao; Cao, Yujin; Zhang, Rubing; Xian, Mo

    2015-01-01

    D-xylonate dehydratase YjhG from Escherichia coli can convert D-xylonate into 2-keto-3-deoxy- D-xylonate (KDX), and is a key enzyme in the biosynthesis of 1,2,4-butanetriol and other chemicals. However, the biochemical properties of YjhG still remain unknown. In this study, the activity assay method for YjhG was established based on semicarbazide method, in which KDX reacts with semicarbazide reagent, and is further quantified by high-resolution mass spectrometry. The effect of reaction conditions on YjhG activity was determined in vitro using purified His-tagged YjhG protein. This enzyme showed maximal activity at 30°C and pH 8.0. Bivalent metal ions such as Mg2+ and Mn2+ activated, whereas Ni2+ and Zn2+ inhibited the activity of YjhG. Under optimal conditions, the Km and Vmax values were 4.88 mM and 78.62 μM l−1h−1, respectively, when using D-xylonate as a substrate. Amino acids sequence alignments and catalytic properties analysis revealed that YjhG might be a member of IlvD/EDD family. Results obtained in this study may lay a foundation for further investigation on YjhG and will benefit its application in biosynthesis of related chemicals. PMID:26083940

  6. ESCRT-0 is not required for ectopic Notch activation and tumor suppression in Drosophila.

    PubMed

    Tognon, Emiliana; Wollscheid, Nadine; Cortese, Katia; Tacchetti, Carlo; Vaccari, Thomas

    2014-01-01

    Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. Here, we report that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, our in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis. PMID:24718108

  7. Transfer of the conjugal tetracycline resistance transposon Tn916 from Streptococcus faecalis to Staphylococcus aureus and identification of some insertion sites in the staphylococcal chromosome.

    PubMed Central

    Jones, J M; Yost, S C; Pattee, P A

    1987-01-01

    The Streptococcus faecalis pheromone-dependent conjugative plasmid pAD1::Tn916 and the membrane filter-dependent conjugative plasmid pPD5::Tn916 were used to introduce Tn916 into Staphylococcus aureus by intergeneric protoplast fusions and intergeneric membrane-filter matings. In recombinants obtained by protoplast fusion where no plasmid DNA could be detected, tetracycline resistance resulted from transposition of Tn916 from pAD1 to the S. aureus chromosome. Transformation analyses showed that S. aureus Tn916 chromosomal insertions occurred near pig, ilv, uraA, tyrB, fus, ala, and the trp operon. DNA hybridization analyses of EcoRI- and HindIII-digested chromosomal DNAs confirmed the diversity of chromosomal sites involved and demonstrated that the inserts were Tn916 insertions rather than integrations of all or part of pAD1::Tn916. Both pAD1::Tn916 and pPD5::Tn916 were transferred to S. aureus by membrane-filter matings. These plasmids remained intact and expressed tetracycline resistance in S. aureus. S. aureus strains carrying pAD1::Tn916, but not a chromosomal insert of Tn916, and any one of several conjugal gentamicin-resistance plasmids lost their ability to serve as conjugal donors of the gentamicin-resistance plasmids. Images PMID:3032908

  8. A SPOPL/Cullin-3 ubiquitin ligase complex regulates endocytic trafficking by targeting EPS15 at endosomes

    PubMed Central

    Gschweitl, Michaela; Ulbricht, Anna; Barnes, Christopher A; Enchev, Radoslav I; Stoffel-Studer, Ingrid; Meyer-Schaller, Nathalie; Huotari, Jatta; Yamauchi, Yohei; Greber, Urs F; Helenius, Ari; Peter, Matthias

    2016-01-01

    Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets. DOI: http://dx.doi.org/10.7554/eLife.13841.001 PMID:27008177

  9. Escherichia coli cell division mutation ftsM1 is in serU

    SciTech Connect

    Leclerc, G.; Sirard, C.; Drapeau, G.R.

    1989-04-01

    The ftsM1 mutation is believed to be in a gene implicated in the regulation of cell division in Escherichia coli because it displayed the lon mutation phenotypes. In this study, we show that this mutation is located in serU, a gene which codes for tRNA(Ser)2, and has the phenotypes of the serU allele supH. Both ftsM1 and supH suppressed the leuB6 and ilvD145 missense mutations, and both conferred temperature and UV light irradiation sensitivity to the harboring cells. Cells which carried the ftsM1 mutation or the supH suppressor had very low colony-forming abilities on salt-free L agar, and this phenotype was almost completely abolished by the presence of plasmids bearing the ftsZ+ gene. Furthermore, sensitivity of the mutant cells to UV irradiation was also markedly diminished when they carried a ftsZ+-bearing plasmid. These results suggest that supH-containing cells have reduced FtsZ activities, in accordance with their displaying the phenotypes of the lon mutant cells. The possibility that ftsM1 (supH) is functionally involved in the biosynthesis of a specific protein which affects cell division is discussed.

  10. Conserved YjgF protein family deaminates reactive enamine/imine intermediates of pyridoxal 5'-phosphate (PLP)-dependent enzyme reactions.

    PubMed

    Lambrecht, Jennifer A; Flynn, Jeffrey M; Downs, Diana M

    2012-01-27

    The YjgF/YER057c/UK114 family of proteins is conserved in all domains of life, suggesting that the role of these proteins arose early and was maintained throughout evolution. Metabolic consequences of lacking this protein in Salmonella enterica and other organisms have been described, but the biochemical function of YjgF remained unknown. This work provides the first description of a conserved biochemical activity for the YjgF protein family. Our data support the conclusion that YjgF proteins have enamine/imine deaminase activity and accelerate the release of ammonia from reactive enamine/imine intermediates of the pyridoxal 5'-phosphate-dependent threonine dehydratase (IlvA). Results from structure-guided mutagenesis experiments suggest that YjgF lacks a catalytic residue and that it facilitates ammonia release by positioning a critical water molecule in the active site. YjgF is renamed RidA (reactive intermediate/imine deaminase A) to reflect the conserved activity of the protein family described here. This study, combined with previous physiological studies on yjgF mutants, suggests that intermediates of pyridoxal 5'-phosphate-mediated reactions may have metabolic consequences in vivo that were previously unappreciated. The conservation of the RidA/YjgF family suggests that reactive enamine/imine metabolites are of concern to all organisms.

  11. Linear and nonlinear ARMA model parameter estimation using an artificial neural network

    NASA Technical Reports Server (NTRS)

    Chon, K. H.; Cohen, R. J.

    1997-01-01

    This paper addresses parametric system identification of linear and nonlinear dynamic systems by analysis of the input and output signals. Specifically, we investigate the relationship between estimation of the system using a feedforward neural network model and estimation of the system by use of linear and nonlinear autoregressive moving-average (ARMA) models. By utilizing a neural network model incorporating a polynomial activation function, we show the equivalence of the artificial neural network to the linear and nonlinear ARMA models. We compare the parameterization of the estimated system using the neural network and ARMA approaches by utilizing data generated by means of computer simulations. Specifically, we show that the parameters of a simulated ARMA system can be obtained from the neural network analysis of the simulated data or by conventional least squares ARMA analysis. The feasibility of applying neural networks with polynomial activation functions to the analysis of experimental data is explored by application to measurements of heart rate (HR) and instantaneous lung volume (ILV) fluctuations.

  12. Metabolic engineering of Candida glabrata for diacetyl production.

    PubMed

    Gao, Xiang; Xu, Nan; Li, Shubo; Liu, Liming

    2014-01-01

    In this study, Candida glabrata, an efficient pyruvate-producing strain, was metabolically engineered for the production of the food ingredient diacetyl. A diacetyl biosynthetic pathway was reconstructed based on genetic modifications and medium optimization. The former included (i) channeling carbon flux into the diacetyl biosynthetic pathway by amplification of acetolactate synthase, (ii) elimination of the branched pathway of α-acetolactate by deleting the ILV5 gene, and (iii) restriction of diacetyl degradation by deleting the BDH gene. The resultant strain showed an almost 1∶1 co-production of α-acetolactate and diacetyl (0.95 g L(-1)). Furthermore, addition of Fe3+ to the medium enhanced the conversion of α-acetolactate to diacetyl and resulted in a two-fold increase in diacetyl production (2.1 g L(-1)). In addition, increased carbon flux was further channeled into diacetyl biosynthetic pathway and a titer of 4.7 g L(-1) of diacetyl was achieved by altering the vitamin level in the flask culture. Thus, this study illustrates that C. glabrata could be tailored as an attractive platform for enhanced biosynthesis of beneficial products from pyruvate by metabolic engineering strategies.

  13. IHF is the limiting host factor in transposition of Pseudomonas putida transposon Tn4652 in stationary phase.

    PubMed

    Ilves, Heili; Hõrak, Rita; Teras, Riho; Kivisaar, Maia

    2004-03-01

    Transpositional activity of mobile elements is not constant. Conditional regulation of host factors involved in transposition may severely change the activity of mobile elements. We have demonstrated previously that transposition of Tn4652 in Pseudomonas putida is a stationary phase-specific event, which requires functional sigma S (Ilves et al., 2001, J Bacteriol 183: 5445-5448). We hypothesized that integration host factor (IHF), the concentration of which is increased in starving P. putida, might contribute to the transposition of Tn4652 as well. Here, we demonstrate that transposition of Tn4652 in stationary phase P. putida is essentially limited by the amount of IHF. No transposition of Tn4652 occurs in a P. putida ihfA-defective strain. Moreover, overexpression of IHF results in significant enhancement of transposition compared with the wild-type strain. This indicates that the amount of IHF is a bottleneck in Tn4652 transposition. Gel mobility shift and DNase I footprinting studies revealed that IHF is necessary for the binding of transposase to both transposon ends. In vitro, transposase can bind to inverted repeats of transposon only after the binding of IHF. The results obtained in this study indicate that, besides sigma S, IHF is another host factor that is implicated in the elevation of transposition in stationary phase. PMID:15009901

  14. Endogenous Synthesis of 2-Aminoacrylate Contributes to Cysteine Sensitivity in Salmonella enterica

    PubMed Central

    Ernst, Dustin C.; Lambrecht, Jennifer A.; Schomer, Rebecca A.

    2014-01-01

    RidA, the archetype member of the widely conserved RidA/YER057c/UK114 family of proteins, prevents reactive enamine/imine intermediates from accumulating in Salmonella enterica by catalyzing their hydrolysis to stable keto acid products. In the absence of RidA, endogenous 2-aminoacrylate persists in the cellular environment long enough to damage a growing list of essential metabolic enzymes. Prior studies have focused on the dehydration of serine by the pyridoxal 5′-phosphate (PLP)-dependent serine/threonine dehydratases, IlvA and TdcB, as sources of endogenous 2-aminoacrylate. The current study describes an additional source of endogenous 2-aminoacrylate derived from cysteine. The results of in vivo analysis show that the cysteine sensitivity of a ridA strain is contingent upon CdsH, the predominant cysteine desulfhydrase in S. enterica. The impact of cysteine on 2-aminoacrylate accumulation is shown to be unaffected by the presence of serine/threonine dehydratases, revealing another mechanism of endogenous 2-aminoacrylate production. Experiments in vitro suggest that 2-aminoacrylate is released from CdsH following cysteine desulfhydration, resulting in an unbound aminoacrylate substrate for RidA. This work expands our understanding of the role played by RidA in preventing enamine stress resulting from multiple normal metabolic processes. PMID:25002544

  15. A SPOPL/Cullin-3 ubiquitin ligase complex regulates endocytic trafficking by targeting EPS15 at endosomes.

    PubMed

    Gschweitl, Michaela; Ulbricht, Anna; Barnes, Christopher A; Enchev, Radoslav I; Stoffel-Studer, Ingrid; Meyer-Schaller, Nathalie; Huotari, Jatta; Yamauchi, Yohei; Greber, Urs F; Helenius, Ari; Peter, Matthias

    2016-01-01

    Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets. PMID:27008177

  16. Inhibition of Bacillus subtilis growth and sporulation by threonine.

    PubMed

    Lamb, D H; Bott, K F

    1979-01-01

    A 1-mg/ml amount of threonine (8.4 mM) inhibited growth and sporulation of Bacillus subtilis 168. Inhibition of sporulation was efficiently reversed by valine and less efficiently by pyruvate, arginine, glutamine, and isoleucine. Inhibition of vegetative growth was reversed by asparate and glutamate as well as by valine, arginine, or glutamine. Cells in minimal growth medium were inhibited only transiently by very high concentrations of threonine, whereas inhibition of sporulation was permanent. Addition of threonine prevented the normal increase in alkaline phosphatase and reduced the production of extracellular protease by about 50%, suggesting that threonine blocked the sporulation process relatively early. 2-Ketobutyrate was able to mimic the effect of threonine on sporulation. Sporulation in a strain selected for resistance to azaleucine was partially resistant. Seventy-five percent of the mutants selected for the ability to grow vegetatively in the presence of high threonine concentrations were found to be simultaneously isoleucine auxotrophs. In at least one of these mutants, the threonine resistance phenotpye could not be dissociated from the isoleucine requirement by transformation. This mutation was closely linked to a known ilvA mutation (recombination index, 0.16). This strain also had reduced intracellular threonine deaminase activity. These results suggest that threonine inhibits B. subtilis by causing valine starvation.

  17. Deep extractive and oxidative desulfurization of dibenzothiophene with C5H9NO·SnCl2 coordinated ionic liquid.

    PubMed

    Li, Fa-tang; Kou, Cheng-guang; Sun, Zhi-min; Hao, Ying-juan; Liu, Rui-hong; Zhao, Di-shun

    2012-02-29

    A new C5H9NO·SnCl2 coordinated ionic liquid (IL) was prepared by reacting N-methyl-pyrrolidone with anhydrous SnCl2. Desulfurization of dibenzothiophene (DBT) via extraction and oxidation with C5H9NO·SnCl2 IL as extractant, H2O2 and equal mol of CH3COOH as oxidants was investigated. The Nernst partition coefficients k(N) of C5H9NO·SnCl2 IL for the DBT in n-octane was above 5.0, showing its excellent extraction ability. During the oxidative desulfurization process, the optimal molar ratio of H2O2/DBT was six. Sulfur removal of DBT in n-octane was 94.8% in 30 min at 30 °C under the conditions of H2O2/DBT molar ratio of six and V (IL):V (oil)=1:3. Moreover, the sulfur removal increased with increasing temperature because of the high reaction rate constant, low viscosity, and high solubility of dibenzothiophene-sulfone in the IL. The kinetics of oxidative desulfurization of DBT was also investigated, and the apparent activation energy was found to be 32.5 kJ/mol. The IL could be recycled six times without a significant decrease in activity. PMID:22230756

  18. LAPTM4B is a PtdIns(4,5)P2 effector that regulates EGFR signaling, lysosomal sorting, and degradation

    PubMed Central

    Tan, Xiaojun; Sun, Yue; Thapa, Narendra; Liao, Yihan; Hedman, Andrew C; Anderson, Richard A

    2015-01-01

    Lysosomal degradation is essential for the termination of EGF-stimulated EGF receptor (EGFR) signaling. This requires EGFR sorting to the intraluminal vesicles (ILVs) of multi-vesicular endosomes (MVEs). Cytosolic proteins including the ESCRT machineries are key regulators of EGFR intraluminal sorting, but roles for endosomal transmembrane proteins in receptor sorting are poorly defined. Here, we show that LAPTM4B, an endosomal transmembrane oncoprotein, inhibits EGF-induced EGFR intraluminal sorting and lysosomal degradation, leading to enhanced and prolonged EGFR signaling. LAPTM4B blocks EGFR sorting by promoting ubiquitination of Hrs (an ESCRT-0 subunit), which inhibits the Hrs association with ubiquitinated EGFR. This is counteracted by the endosomal PIP kinase, PIPKIγi5, which directly binds LAPTM4B and neutralizes the inhibitory function of LAPTM4B in EGFR sorting by generating PtdIns(4,5)P2 and recruiting SNX5. PtdIns(4,5)P2 and SNX5 function together to protect Hrs from ubiquitination, thereby promoting EGFR intraluminal sorting. These results reveal an essential layer of EGFR trafficking regulated by LAPTM4B, PtdIns(4,5)P2 signaling, and the ESCRT complex and define a mechanism by which the oncoprotein LAPTM4B can transform cells and promote tumor progression. PMID:25588945

  19. Proteomic analysis of cell wall in four pathogenic species of Candida exposed to oxidative stress.

    PubMed

    Ramírez-Quijas, Mayra Denisse; López-Romero, Everardo; Cuéllar-Cruz, Mayra

    2015-10-01

    In order for Candida species to adhere and colonize human host cells they must express cell wall proteins (CWP) and adapt to reactive oxygen species (ROS) generated by phagocytic cells of the human host during the respiratory burst. However, how these pathogens change the expression of CWP in response to oxidative stress (OSR) is not known. Here, fifteen moonlight-like CWP were identified that expressed differentially in four species of Candida after they were exposed to H2O2 or menadione (O2(-)). These proteins included: (i) glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (Gapdh), fructose-bisphosphate aldolase (Fba1), phosphoglycerate mutase (Gpm1), phosphoglycerate kinase (Pgk), pyruvate kinase (Pk) and enolase (Eno1); (ii) the heat shock proteins Ssb1 and Ssa2; (iii) OSR proteins such as peroxyredoxin (Tsa1), the stress protein Ddr48 (Ddr48) and glutathione reductase (Glr1); (iv) other metabolic enzymes such as ketol-acid reductoisomerase (Ilv5) and pyruvate decarboxylase (Pdc1); and (v) other proteins such as elongation factor 1-beta (Efb1) and the 14-3-3 protein homolog. RT-PCR revealed that transcription of the genes coding for some of the identified CWP are differentially regulated. To our knowledge this is the first report showing that moonlight-like CWP are the first line of defense of Candida against ROS, and that they are differentially regulated in each of these pathogens.

  20. De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth

    PubMed Central

    Fisher, Michael A.; McKinley, Kara L.; Bradley, Luke H.; Viola, Sara R.; Hecht, Michael H.

    2011-01-01

    A central challenge of synthetic biology is to enable the growth of living systems using parts that are not derived from nature, but designed and synthesized in the laboratory. As an initial step toward achieving this goal, we probed the ability of a collection of >106 de novo designed proteins to provide biological functions necessary to sustain cell growth. Our collection of proteins was drawn from a combinatorial library of 102-residue sequences, designed by binary patterning of polar and nonpolar residues to fold into stable 4-helix bundles. We probed the capacity of proteins from this library to function in vivo by testing their abilities to rescue 27 different knockout strains of Escherichia coli, each deleted for a conditionally essential gene. Four different strains – ΔserB, ΔgltA, ΔilvA, and Δfes – were rescued by specific sequences from our library. Further experiments demonstrated that a strain simultaneously deleted for all four genes was rescued by co-expression of four novel sequences. Thus, cells deleted for ∼0.1% of the E. coli genome (and ∼1% of the genes required for growth under nutrient-poor conditions) can be sustained by sequences designed de novo. PMID:21245923

  1. Multilocus sequence typing of Campylobacter concisus from Danish diarrheic patients.

    PubMed

    Nielsen, Hans Linde; Nielsen, Henrik; Torpdahl, Mia

    2016-01-01

    The emerging enteric pathogen Campylobacter concisus is associated with prolonged diarrhea and inflammatory bowel disease. Previous studies have shown that C. concisus strains are very genetically diverse. Nevertheless, C. concisus strains have been divided into two genomospecies, where GS1 strains have been isolated predominantly from healthy individuals, while the GS2 cluster consists of isolates primarily from diarrheic individuals. The aim of the present study was to determine the genetic diversity of C. concisus isolates from Danish diarrheic patients. Multilocus sequence typing using the loci aspA, atpA, glnA, gltA, glyA, ilvD and pgm, as well as genomospecies based on specific differences in the 23S rRNA, was used to characterize 67 isolates (63 fecal and 4 oral), from 49 patients with different clinical presentations (29 with diarrhea, eight with bloody diarrhea, seven with collagenous colitis and five with Crohn's disease). MLST revealed a high diversity of C. concisus with 53 sequence types (STs), of which 52 were identified as 'new' STs. Allele sequences showed more than 90 % similarity between isolates, with only four outliers. Dendrogram profiles of each allele showed a division into two groups, which more or less correlated with genomospecies A and genomospecies B. However, in contrary to previous results, this subgrouping had no association to the clinical severity of disease.

  2. Reduction of the off-flavor volatile generated by the yogurt starter culture including Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in soymilk.

    PubMed

    Kaneko, Daisuke; Igarashi, Toshinori; Aoyama, Kenji

    2014-02-19

    Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus establish a symbiotic relationship in milk; however, S. thermophilus predominantly grows in soymilk. This study determined that excess diacetyl was notably generated mainly by S. thermophilus in soymilk, and this flavor compound created an unpleasant odor in fermented soymilk. The addition of l-valine to soymilk reduced the amount of diacetyl and increased the levels of acetoin during fermentation by S. thermophilus . In addition, it was found that the expression of the ilvC gene was repressed and that of the als and aldB genes was stimulated in S. thermophilus by l-valine. Sensory evaluations with the triangle difference test and a preference test showed that the soymilk fermented with l-valine was significantly preferred compared with that without l-valine. In this study, we successfully controlled the metabolic flux of S. thermophilus in soymilk and produced more favorable fermented soymilk without the use of genetically modified lactic acid bacteria strains.

  3. Complete Plastid Genome Sequence of the Brown Alga Undaria pinnatifida

    PubMed Central

    Liu, Tao; Wang, Guoliang; Chi, Shan; Liu, Cui; Wang, Haiyang

    2015-01-01

    In this study, we fully sequenced the circular plastid genome of a brown alga, Undaria pinnatifida. The genome is 130,383 base pairs (bp) in size; it contains a large single-copy (LSC, 76,598 bp) and a small single-copy region (SSC, 42,977 bp), separated by two inverted repeats (IRa and IRb: 5,404 bp). The genome contains 139 protein-coding, 28 tRNA, and 6 rRNA genes; none of these genes contains introns. Organization and gene contents of the U. pinnatifida plastid genome were similar to those of Saccharina japonica. There is a co-linear relationship between the plastid genome of U. pinnatifida and that of three previously sequenced large brown algal species. Phylogenetic analyses of 43 taxa based on 23 plastid protein-coding genes grouped all plastids into a red or green lineage. In the large brown algae branch, U. pinnatifida and S. japonica formed a sister clade with much closer relationship to Ectocarpus siliculosus than to Fucus vesiculosus. For the first time, the start codon ATT was identified in the plastid genome of large brown algae, in the atpA gene of U. pinnatifida. In addition, we found a gene-length change induced by a 3-bp repetitive DNA in ycf35 and ilvB genes of the U. pinnatifida plastid genome. PMID:26426800

  4. Metabolic Engineering of Candida glabrata for Diacetyl Production

    PubMed Central

    Gao, Xiang; Xu, Nan; Li, Shubo; Liu, Liming

    2014-01-01

    In this study, Candida glabrata, an efficient pyruvate-producing strain, was metabolically engineered for the production of the food ingredient diacetyl. A diacetyl biosynthetic pathway was reconstructed based on genetic modifications and medium optimization. The former included (i) channeling carbon flux into the diacetyl biosynthetic pathway by amplification of acetolactate synthase, (ii) elimination of the branched pathway of α-acetolactate by deleting the ILV5 gene, and (iii) restriction of diacetyl degradation by deleting the BDH gene. The resultant strain showed an almost 1∶1 co-production of α-acetolactate and diacetyl (0.95 g L−1). Furthermore, addition of Fe3+ to the medium enhanced the conversion of α-acetolactate to diacetyl and resulted in a two-fold increase in diacetyl production (2.1 g L−1). In addition, increased carbon flux was further channeled into diacetyl biosynthetic pathway and a titer of 4.7 g L−1 of diacetyl was achieved by altering the vitamin level in the flask culture. Thus, this study illustrates that C. glabrata could be tailored as an attractive platform for enhanced biosynthesis of beneficial products from pyruvate by metabolic engineering strategies. PMID:24614328

  5. Candida albicans PROTEIN PROFILE CHANGES IN RESPONSE TO THE BUTANOLIC EXTRACT OF Sapindus saponariaL.

    PubMed Central

    FIORINI, Adriana; ROSADO, Fabio Rogério; BETTEGA, Eliane Martins da Silva; MELO, Kátia Cristina Sibin; KUKOLJ, Caroline; BONFIM-MENDONÇA, Patrícia de Souza; SHINOBU-MESQUITA, Cristiane Suemi; GHIRALDI, Luciana Dias; CAMPANERUT, Paula Aline Zanetti; CAPOCI, Isis Regina Grenier; GODOY, Janine Silva Ribeiro; FERREIRA, Izabel Cristina Piloto; SVIDZINSKI, Terezinha Inez Estivalet

    2016-01-01

    Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC) and sub-minimal inhibitory concentration (sub-MIC) of the butanolic extract (BUTE) of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1), amino acid metabolism (ILV5, PDC11) and protein synthesis (ASC1) pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria(e.g., saponins and sesquiterpene oligoglycosides), it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds. PMID:27074319

  6. Bacillithiol has a role in Fe-S cluster biogenesis in Staphylococcus aureus

    PubMed Central

    Rosario-Cruz, Zuelay; Chahal, Harsimranjit K.; Mike, Laura A.; Skaar, Eric P.; Boyd, Jeffrey M.

    2015-01-01

    Summary Staphylococcus aureus does not produce the low-molecular-weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH-deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activity of the iron-sulfur (Fe-S) cluster-dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH-deficient cells also had decreased aconitase and glutamate synthase activities suggesting a general defect in Fe-S cluster biogenesis. The phenotypes of the BSH-deficient strains were exacerbated in strains lacking the Fe-S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu suggesting functional overlap between BSH and Fe-S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe-S clusters to apo-aconitase verifying that it serves as an Fe-S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe-S clusters to apo-proteins in S. aureus. PMID:26135358

  7. Recombination-induced suppression of cell division following P1-mediated generalized transduction in Klebsiella aerogenes.

    PubMed

    Bender, R A; Sambucetti, L C

    1983-01-01

    Klebsiella aerogenes recombinants resulting from bacteriophage P1-mediated generalized transduction failed to increase in number for approximately six generations after transduction. Nevertheless these recombinants continued to grow and became sensitive to penicillin after a transient resistance, suggesting that the cells were growing as long, non-dividing filaments. When filamentous cells were isolated from transduced cultures by gradient centrifugation, recombinants were 1000-fold more frequent among the filaments than among the normal-sized cells. The suppression of cell-division lasted for six generations whether markers near the origin (gln, ilv) or terminus (his, trp) of chromosome replication were used, despite a 50-fold difference in transduction frequencies for these markers. The suppression of cell division was a host response to recombination rather than to P1 invasion since cells lysogenized by P1 in these same experiments showed only a short (two generation) suppression of cell division. We speculate that the suppression of cell-division is an SOS response triggered by the degraded DNA not incorporated in the final recombinant. We demonstrate that both the filamentation and the transient penicillin resistance of recombinant cells can be exploited to enrich greatly for recombinants, raising transduction frequencies to as high as 10(-3).

  8. One-step fermentative production of poly(lactate-co-glycolate) from carbohydrates in Escherichia coli.

    PubMed

    Choi, So Young; Park, Si Jae; Kim, Won Jun; Yang, Jung Eun; Lee, Hyuk; Shin, Jihoon; Lee, Sang Yup

    2016-04-01

    Poly(lactate-co-glycolate) (PLGA) is a widely used biodegradable and biocompatible synthetic polymer. Here we report one-step fermentative production of PLGA in engineered Escherichia coli harboring an evolved polyhydroxyalkanoate (PHA) synthase that polymerizes D-lactyl-CoA and glycolyl-CoA into PLGA. Introduction of the Dahms pathway enables production of glycolate from xylose. Deletion of ptsG enables simultaneous utilization of glucose and xylose. An evolved propionyl-CoA transferase converts D-lactate and glycolate to D-lactyl-CoA and glycolyl-CoA, respectively. Deletion of adhE, frdB, pflB and poxB prevents by-product formation. We also demonstrate modulation of the monomer fractions in PLGA by overexpressing ldhA and deleting dld to increase the proportion of D-lactate or by deleting aceB, glcB, glcD, glcE, glcF and glcG to increase the proportion of glycolate. Incorporation of 2-hydroxybutyrate is prevented by deleting ilvA or feeding strains with L-isoleucine. The utility of our approach for generating diverse forms of PLGA is shown by the production of copolymers containing 3-hydroxybutyrate, 4-hydroxybutyrate or 2-hydroxyisovalerate. PMID:26950748

  9. Complete primary structure of the triple-helical region and the carboxyl-terminal domain of a new type IV collagen chain, alpha 5(IV).

    PubMed

    Pihlajaniemi, T; Pohjolainen, E R; Myers, J C

    1990-08-15

    .-R., Kadri, A.S., Goddard, A.D., Sheer, D., Solomon, E., and Pihlajaniemi, T. (1990) Am. J. Hum. Genet. 46, 1024-1033). Consequently, the newly discovered alpha 5(IV) collagen chain may have a critical role in inherited diseases of connective tissue. PMID:2380186

  10. Triad Resonance in the Gravity-Acoustic Family

    NASA Astrophysics Data System (ADS)

    Kadri, U.

    2015-12-01

    Resonance interactions of waves play a prominent role in energy share among the different wave types involved. Such interactions may significantly contribute, among others, to the evolution of the ocean energy spectrum by exchanging energy between surface-gravity waves; surface and internal gravity waves; or even surface and compression-type waves, that can transfer energy from the upper ocean through the whole water column reaching down to the seafloor. A resonant triad occurs among a triplet of waves, usually involving interaction of nonlinear terms of second order perturbed equations. Until recently, it has been believed that in a homogeneous fluid a resonant triad is possible only when tension forces are included, or at the limit of a shallow water, and that when the compressibility of water is considered, no resonant triads can occur within the family of gravity-acoustic waves. However, more recently it has been proved that, under some circumstances, resonant triads comprising two opposing surface-gravity waves of similar periods (though not identical) and a much longer acoustic-gravity wave, of almost double the frequency, exist [Kadri and Stiassnie 2013, J. Fluid Mech.735 R6]. Here, I report on a new resonant triad involving a gravity wave and two acoustic waves of almost double the length. Interestingly, the two acoustic waves propagate in the same direction with similar wavelengths, that are almost double of that of the gravity wave. The evolution of the wave triad amplitudes is periodic and it is derived analytically, in terms of Jacobian elliptic functions and elliptic integrals. The physical importance of this type of triad interactions is the modulation of pertinent acoustic signals, leading to inaccurate signal perceptions. Enclosed figure: presents an example spatio-temporal evolution of the wave triad amplitudes. The gravity wave (top) remains almost unaltered, while the envelope slowly displaces to the left. However, the prescribed acoustic

  11. The Cpx proteins of Escherichia coli K-12: evidence that cpxA, ecfB, ssd, and eup mutations all identify the same gene.

    PubMed

    Rainwater, S; Silverman, P M

    1990-05-01

    An existing cpxA(Ts) mutant was resistant to amikacin at levels that inhibited completely the growth of a cpxA+ and a cpxA deletion strain and failed to grow as efficiently on exogenous proline. These properties are similar to those of mutants altered in a gene mapped to the cpxA locus and variously designated as ecfB, ssd, and eup. The amikacin resistance phenotype of the cpxA mutant was inseparable by recombination from the cpxA mutant phenotype (inability to grow at 41 degrees C without exogenous isoleucine and valine) and was recessive to the cpxA+ allele of a recombinant plasmid. Using methods that ensured independent mutations in the cpxA region of the chromosome, we isolated six new amikacin-resistant mutants following nitrosoguanidine mutagenesis. Three-factor crosses mapped the mutations to the cpxA locus. When transferred by P1 transduction to a cpxB11 Hfr strain, each of the mutations conferred the Tra- and Ilv- phenotypes characteristic of earlier cpxA mutants. Two of the new mutations led to a significantly impaired ability to utilize exogenous proline, and four led to partial resistance to colicin A. Two of the new cpxA alleles were recessive to the cpxA+ allele, and four were dominant, albeit to different degrees. On the basis of these data, we argue that cpxA, ecfB, eup, and ssd are all the same gene. We discuss the cellular function of the cpxA gene product in that light.

  12. Transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in Corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis.

    PubMed

    Neshat, Armin; Mentz, Almut; Rückert, Christian; Kalinowski, Jörn

    2014-11-20

    The Gram-positive bacterium Corynebacterium glutamicum belongs to the order Corynebacteriales and is used as a producer of amino acids at industrial scales. Due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. Applying the high-resolution technique of transcriptome sequencing (RNA-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the C. glutamicum transcriptome. By analyzing RNA-seq data from a small RNA cDNA library of C. glutamicum, short transcripts in the known transcriptional attenuators sites of the trp operon, the ilvBNC operon and the leuA gene were verified. Furthermore, whole transcriptome RNA-seq data were used to elucidate the transcriptional organization of these three amino acid biosynthesis operons. In addition, we discovered and analyzed the novel attenuator aroR, located upstream of the aroF gene (cg1129). The DAHP synthase encoded by aroF catalyzes the first step in aromatic amino acid synthesis. The AroR leader peptide contains the amino acid sequence motif F-Y-F, indicating a regulatory effect by phenylalanine and tyrosine. Analysis by real-time RT-PCR suggests that the attenuator regulates the transcription of aroF in dependence of the cellular amount of tRNA loaded with phenylalanine when comparing a phenylalanine-auxotrophic C. glutamicum mutant fed with limiting and excess amounts of a phenylalanine-containing dipeptide. Additionally, the very interesting finding was made that all analyzed attenuators are leaderless transcripts. PMID:24910972

  13. Photographing Internal Fractures of the Archaeological Statues with 3D Visualization of Ground Penetrating Radar Data

    NASA Astrophysics Data System (ADS)

    Kadioglu, S.; Kadioglu, Y. K.

    2009-04-01

    PHOTOGRAPHING INTERNAL FRACTURES OF THE ARCHAEOLOGICAL STATUES WITH 3D VISUALIZATION OF GROUND PENETRATING RADAR DATA Selma KADIOGLU1 and Yusuf K. KADIOGLU2 1Ankara University, Faculty of Engineering, Department of Geophysical Engineering, 06100 Tandogan/ANKARA-TURKEY kadioglu@eng.ankara.edu.tr 2Ankara University, Faculty of Engineering, Department of Geological Engineering, 06100 Tandogan/ANKARA-TURKEY kadi@eng.ankara.edu.tr The aim of the study is to illustrate a new approach to image the discontinuities in the archaeological statues before restoration studies using ground penetrating radar (GPR) method. The method was successfully applied to detect and map the fractures and cavities of the two monument groups and lion statues in Mustafa Kemal ATATURK's tumb (ANITKABIR) in Ankara-Turkey. The tumb, which has been started to build in 1944 and completed in 1953, represents Turkish people and Ataturk, who is founder of the Republic of Turkey. Therefore this monument is very important for Turkish people. The monument groups and lion statues have been built from travertine rocks. These travertine have vesicular textures with the percent of 12. They have been mainly composed of calcite, aragonite with rare amount of plant relict and clay minerals. The concentrations of Fe, Mg, Cl and Mn may lead to verify their colours changing from white through pale green to beige. The atmospheric contamination of Ankara has been caused to cover some parts of the surface of these travertine with a thin film of Pb as blackish in colour. The micro fractures have been observed specially at the rim of the vesicular of the rocks by the polarizing microscope. Parallel two dimensional (2D) GPR profile data with 10cm profile space were acquired by RAMAC CU II system with 1600 MHz shielded antenna on the monument groups (three women, three men and 24 lion statues) and then a three dimensional (3D) data volume were built using parallel 2D GPR data. Air-filled fractures and cavities in the

  14. 40Ar/39Ar Dating of Zn-Pb-Ag Mineralization in the Northern Brooks Range, Alaska

    USGS Publications Warehouse

    Werdon, Melanie B.; Layer, Paul W.; Newberry, Rainer J.

    2004-01-01

    The 40Ar/39Ar laser step-heating method potentially can be used to provide absolute ages for a number of formerly undatable, low-temperature ore deposits. This study demonstrates the use of this method by determining absolute ages for Zn-Pb-Ag sediment-hosted massive sulfide deposits and vein-breccia occurrences found throughout a 300-km-long, east-west-trending belt in the northern Brooks Range, Alaska. Massive sulfide deposits are hosted by Mississippian to Pennsylvanian(?) black carbonaceous shale, siliceous mudstone, and lesser chert and carbonate turbidites of the Kuna Formation (e.g., Red Dog, Anarraaq, Lik (Su), and Drenchwater). The vein-breccia occurrences (e.g., Husky, Story Creek, West Kivliktort Mountain, Vidlee, and Kady) are hosted by a deformed but only weakly metamorphosed package of Upper Devonian to Lower Mississippian mixed continental and marine clastic rocks (the Endicott Group) that stratigraphically underlie the Kuna Formation. The vein-breccias are mineralogically similar to, but not spatially associated with, known massive sulfide deposits. The region's largest shale-hosted massive sulfide deposit is Red Dog; it has reserves of 148 Mt grading 16.6 percent zinc, 4.5 percent lead, and 77 g of silver per tonne. Hydrothermally produced white mica in a whole-rock sample from a sulfide-bearing igneous sill within the Red Dog deposit yielded a plateau age of 314.5 Ma. The plateau age of this whole-rock sample records the time at which temperatures cooled below the argon closure temperature of the white mica and is interpreted to represent the minimum age limit for massive sulfide-related hydrothermal activity in the Red Dog deposit. Sulfide-bearing quartz veins at Drenchwater crosscut a hypabyssal intrusion with a maximum biotite age of 337.0 Ma. Despite relatively low sulfide deposition temperatures in the vein-breccia occurrences (162°-251°C), detrital white mica in sandstone immediately adjacent to large vein-breccia zones was partially to

  15. Evaluation of atazanavir Ctrough, atazanavir genotypic inhibitory quotient, and baseline HIV genotype as predictors of a 24-week virological response in highly drug-experienced, HIV-infected patients treated with unboosted atazanavir.

    PubMed

    Gianotti, Nicola; Seminari, Elena; Guffanti, Monica; Boeri, Enzo; Villani, Paola; Regazzi, Mario; Bigoloni, Alba; Schira, Giulia; Tiberi, Simon; Fusetti, Giuliana; Lazzarin, Adriano; Castagna, Antonella

    2005-04-01

    The objective of this study was to evaluate virological and pharmacological determinants of a 24-week virological response to unboosted atazanavir (ATV) in highly drug-experienced HIV-infected patients. Among patients enrolled in the ATV Expanded Access Program, those with HIV-RNA >1000 copies/mL, a genotype performed within three months from the baseline (BL), and who completed 24 weeks of treatment, were included. They received at least three antiretrovirals, including ATV 400 mg once daily without boosting. ATV plasma levels were evaluated after four weeks of treatment by high performance liquid chromatography (HPLC). ATV genotypic inhibitory quotient (GIQ) was calculated as the ratio between ATV Ctrough and the number of the BL ATV-related protease resistance mutations (among the following: 10I/V/F, 20R/M/I, 24I, 331/F/V, 36I/L/V, 46I/L, 48V, 54V/L, 63P, 71V/T/I, 73C/S/T/A, 82A/F/S/T, 84V, and 90M). Thirty-five subjects were included. At baseline, median (interquartile range) CD4+ T-lymphocytes, HIV-RNA, and ATV resistance mutations were 232.5 (106-303)/microL, 4.7 (4.2-5.1) log10 copies/mL, 2 (1-6), respectively. Thirteen (37.1%) subjects were off-therapy and 11 (31.4%) showed no PI mutation at baseline. Median steady-state ATV Ctrough was 230 ng/mL (87-520), for an ATV GIQ of 86.5 (25.5-165.5). Median HIV-RNA changes from baseline at weeks 4, 12 and 24 were -1.76 (from -0.44 to -2.12), -1.41 (from -0.41 to -2.81) and -1.44 (from -0.42 to -2.71) log10, respectively. The HIV-RNA changes were correlated to the number of ATV resistance mutations at each time point (P < 0.05), whereas no correlation was found between ATV Ctrough or ATV GIQ and HIV-RNA changes. In conclusion, the number of ATV resistance mutations is the only correlate to virological response through 24 weeks of treatment with unboosted atazanavir 400 mg once daily.

  16. Use of the valine biosynthetic pathway to convert glucose into isobutanol.

    PubMed

    Savrasova, Ekaterina A; Kivero, Aleksander D; Shakulov, Rustem S; Stoynova, Nataliya V

    2011-09-01

    Microbiological synthesis of higher alcohols (1-butanol, isobutanol, 2-methyl-1-butanol, etc.) from plant biomass is critically important due to their advantages over ethanol as a motor fuel. In recent years, the use of branched-chain amino acid (BCAA) biosynthesis pathways together with heterologous Ehrlich pathway enzyme system (Hazelwood et al. in Appl Environ Microbiol 74:2259-2266, 2008) has been proposed by the Liao group as an alternative approach to aerobic production of higher alcohols as new-generation biofuels (Atsumi et al. in Nature 451:86-90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89-98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769-5775, 2008; Shen and Liao in Metab Eng 10:312-320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471-479, 2009). On the basis of these remarkable investigations, we re-engineered Escherichia coli valine-producing strain H-81, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar into isobutanol. To redirect valine biosynthesis to the production of alcohol, we also--as has been demonstrated previously (Atsumi et al. in Nature 451:86-90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89-98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769-5775, 2008; Shen and Liao in Metab Eng 10:312-320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471-479, 2009)--used enzymes of Ehrlich pathway. In particular, in our study, the following heterologous proteins were exploited: branched-chain 2-keto acid decarboxylase (BCKAD) encoded by the kdcA gene from Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by the ADH2 gene from Saccharomyces cerevisiae. We show that expression of both of these genes in the valine-producing strain H-81 results in accumulation of isobutanol instead of valine. Expression of BCKAD

  17. Crystallization and X-ray diffraction analysis of an L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and a D-xylonate dehydratase from Caulobacter crescentus.

    PubMed

    Rahman, Mohammad Mubinur; Andberg, Martina; Koivula, Anu; Rouvinen, Juha; Hakulinen, Nina

    2016-08-01

    L-Arabinonate dehydratase (EC 4.2.1.25) and D-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. L-Arabinonate dehydratase converts L-arabinonate into 2-dehydro-3-deoxy-L-arabinonate, and D-xylonate dehydratase catalyzes the dehydration of D-xylonate to 2-dehydro-3-deoxy-D-xylonate. L-Arabinonate and D-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any L-arabinonate or D-xylonate dehydratase is available in the PDB. In this study, recombinant L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT) and D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) were heterologously expressed in Escherichia coli and purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals of RlArDHT that diffracted to 2.40 Å resolution were obtained using sodium formate as a precipitating agent. They belonged to space group P21, with unit-cell parameters a = 106.07, b = 208.61, c = 147.09 Å, β = 90.43°. Eight RlArDHT molecules (two tetramers) in the asymmetric unit give a VM value of 3.2 Å(3) Da(-1) and a solvent content of 62%. Crystals of CcXyDHT that diffracted to 2.66 Å resolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 Å, β = 97.38°. Four CcXyDHT molecules (a tetramer) in the asymmetric unit give a VM value of 4.0 Å(3) Da(-1) and a solvent content of 69%. PMID:27487924

  18. Geochemical Data Package for Performance Assessment Calculations Related to the Savannah River Site

    SciTech Connect

    Kaplan, D

    2006-02-28

    The Savannah River Site disposes of certain types of radioactive waste within subsurface-engineered facilities. One of the tools used to establish the capacity of a given site to safely store radioactive waste (i.e., that a site does not exceed its Waste Acceptance Criteria) is the Performance Assessment (PA). The objective of this document is to provide the geochemical values for the PA calculations. This work is being conducted as part of the on-going maintenance program that permits the PA to periodically update existing calculations when new data becomes available. Because application of values without full understanding of their original purpose may lead to misuse, this document also provides the geochemical conceptual model, approach used for selecting the values, the justification for selecting data, and the assumptions made to assure that the conceptual and numerical geochemical models are reasonably conservative (i.e., reflect conditions that will tend to predict the maximum risk to the hypothetical recipient). The geochemical parameters describe transport processes for 38 elements (>90 radioisotopes) potentially occurring within eight disposal units (Slit Trenches, Engineered Trenches, Low Activity Waste (LAW) Vault, Intermediate Level (ILV) Vaults, TRU-Pad-1, Naval Reactor Waste Pads, Components-in-Grout Trenches, and Saltstone Facility). This work builds upon well-documented work from previous PA calculations (McDowell-Boyer et al. 2000). The new geochemical concepts introduced in this data package are: (1) In the past, solubility products were used only in a few conditions (element existing in a specific environmental setting). This has been expanded to >100 conditions. (2) Radionuclide chemistry in cementitious environments is described through the use of both the Kd and apparent solubility concentration limit. Furthermore, the solid phase is assumed to age during the assessment period (thousands of years), resulting in three main types of controlling

  19. Crystallization and X-ray diffraction analysis of an l-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and a d-xylonate dehydratase from Caulobacter crescentus

    PubMed Central

    Rahman, Mohammad Mubinur; Andberg, Martina; Koivula, Anu; Rouvinen, Juha; Hakulinen, Nina

    2016-01-01

    l-Arabinonate dehydratase (EC 4.2.1.25) and d-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. l-Arabinonate dehydratase converts l-arabinonate into 2-dehydro-3-deoxy-l-arabinonate, and d-xylonate dehydratase catalyzes the dehydration of d-xylonate to 2-dehydro-3-deoxy-d-xylonate. l-Arabinonate and d-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any l-arabinonate or d-xylonate dehydratase is available in the PDB. In this study, recombinant l-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT) and d-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) were heterologously expressed in Escherichia coli and purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals of RlArDHT that diffracted to 2.40 Å resolution were obtained using sodium formate as a precipitating agent. They belonged to space group P21, with unit-cell parameters a = 106.07, b = 208.61, c = 147.09 Å, β = 90.43°. Eight RlArDHT molecules (two tetramers) in the asymmetric unit give a V M value of 3.2 Å3 Da−1 and a solvent content of 62%. Crystals of CcXyDHT that diffracted to 2.66 Å resolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 Å, β = 97.38°. Four CcXyDHT molecules (a tetramer) in the asymmetric unit give a V M value of 4.0 Å3 Da−1 and a solvent content of 69%. PMID:27487924

  20. Comparative analysis of cyanobacterial superoxide dismutases to discriminate canonical forms

    PubMed Central

    Priya, Balakrishnan; Premanandh, Jagadeesan; Dhanalakshmi, Raman T; Seethalakshmi, Thangaraj; Uma, Lakshmanan; Prabaharan, Dharmar; Subramanian, Gopalakrishnan

    2007-01-01

    Background Superoxide dismutases (SOD) are ubiquitous metalloenzymes that catalyze the disproportion of superoxide to peroxide and molecular oxygen through alternate oxidation and reduction of their metal ions. In general, SODs are classified into four forms by their catalytic metals namely; FeSOD, MnSOD, Cu/ZnSOD and NiSOD. In addition, a cambialistic form that uses Fe/Mn in its active site also exists. Cyanobacteria, the oxygen evolving photosynthetic prokaryotes, produce reactive oxygen species that can damage cellular components leading to cell death. Thus, the co-evolution of an antioxidant system was necessary for the survival of photosynthetic organisms with SOD as the initial enzyme evolved to alleviate the toxic effect. Cyanobacteria represent the first oxygenic photoautotrophs and their SOD sequences available in the databases lack clear annotation. Hence, the present study focuses on structure and sequence pattern of subsets of cyanobacterial superoxide dismutases. Result The sequence conservation and structural analysis of Fe (Thermosynechococcus elongatus BP1) and MnSOD (Anabaena sp. PCC7120) reveal the sharing of N and C terminal domains. At the C terminal domain, the metal binding motif in cyanoprokaryotes is DVWEHAYY while it is D-X-[WF]-E-H-[STA]-[FY]-[FY] in other pro- and eukaryotes. The cyanobacterial FeSOD differs from MnSOD at least in three ways viz. (i) FeSOD has a metal specific signature F184X3A188Q189.......T280......F/Y303 while, in Mn it is R184X3G188G189......G280......W303, (ii) aspartate ligand forms a hydrogen bond from the active site with the outer sphere residue of W243 in Fe where as it is Q262 in MnSOD; and (iii) two unique lysine residues at positions 201 and 255 with a photosynthetic role, found only in FeSOD. Further, most of the cyanobacterial Mn metalloforms have a specific transmembrane hydrophobic pocket that distinguishes FeSOD from Mn isoform. Cyanobacterial Cu/ZnSOD has a copper domain and two different signatures G-F-H-[ILV

  1. Global expression profiling of Bacillus subtilis cells during industrial-close fed-batch fermentations with different nitrogen sources.

    PubMed

    Jürgen, Britta; Tobisch, Steffen; Wümpelmann, Mogens; Gördes, Dirk; Koch, Andreas; Thurow, Kerstin; Albrecht, Dirk; Hecker, Michael; Schweder, Thomas

    2005-11-01

    A detailed gene expression analysis of industrial-close Bacillus subtilis fed-batch fermentation processes with casamino acids as the only nitrogen source and with a reduced casamino acid concentration but supplemented by ammonia was carried out. Although glutamine and arginine are supposed to be the preferred nitrogen sources of B. subtilis, we demonstrate that a combined feeding of ammonia and casamino acids supports cell growth under fed-batch fermentation conditions. The transcriptome and proteome analyses revealed that the additional feeding of ammonia in combination with a reduced amino acid concentration results in a significantly lower expression level of the glnAR or tnrA genes, coding for proteins, which are mainly involved in the nitrogen metabolism of B. subtilis. However, the mRNA levels of the genes of the ilvBHC-leuABD and hom-thrCB operons were significantly increased, indicating a valine, leucine, isoleucine, and threonine limitation under these fermentation conditions. In contrast, during the fermentation with casamino acids as the only nitrogen source, several genes, which play a crucial role in nitrogen metabolism of B. subtilis (e.g., glnAR, nasCDE, nrgAB, and ureABC), were up-regulated, indicating a nitrogen limitation under these conditions. Furthermore, increased expression of genes, which are involved in motility and chemotaxis (e.g., hag, fliT) and in acetoin metabolism (e.g., acoABCL), was determined during the fermentation with the mixed nitrogen source of casamino acids and ammonia, indicating a carbon limitation under these fermentation conditions. Under high cell density and slow growth rate conditions a weak up-regulation of autolysis genes could be observed as well as the induction of a number of genes involved in motility, chemotaxis and general stress response. Results of this study allowed the selection of marker genes, which could be used for the monitoring of B. subtilis fermentation processes. The data suggest for example aco

  2. Development of a multi-locus sequence typing scheme for Laribacter hongkongensis, a novel bacterium associated with freshwater fish-borne gastroenteritis and traveler's diarrhea

    PubMed Central

    2009-01-01

    Background Laribacter hongkongensis is a newly discovered, facultative anaerobic, Gram-negative, motile, sea gull-shaped rod associated with freshwater fish borne gastroenteritis and traveler's diarrhea. A highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of L. hongkongensis. In this study, a multilocus sequence typing (MLST) system was developed for L. hongkongensis. The system was used to characterize 146 L. hongkongensis isolates, including 39 from humans and 107 from fish. Results Fragments (362 to 504 bp) of seven housekeeping genes were amplified and sequenced. Among the 3068 bp of the seven loci, 332 polymorphic sites were observed. The median number of alleles at each locus was 34 [range 22 (ilvC) to 45 (thiC)]. All seven genes showed very low dn/ds ratios of < 0.04, indicating that no strong positive selective pressure is present. A total of 97 different sequence types (STs) were assigned to the 146 isolates, with 80 STs identified only once. The overall discriminatory power was 0.9861. eBURST grouped the isolates into 12 lineages, with six groups containing only isolates from fish and three groups only isolates from humans. Standardized index of association (ISA) measurement showed significant linkage disequilibrium in isolates from both humans and fish. The ISA for the isolates from humans and fish were 0.270 and 0.636, indicating the isolates from fish were more clonal than the isolates from humans. Only one interconnected network (acnB) was detected in the split graphs. The P-value (P = 0) of sum of the squares of condensed fragments in Sawyer's test showed evidence of intragenic recombination in the rho, acnB and thiC loci, but the P-value (P = 1) of maximum condensed fragment in these gene loci did not show evidence of intragenic recombination. Congruence analysis showed that all the pairwise comparisons of the 7 MLST loci were incongruent, indicating that recombination played a substantial

  3. Avibactam and Inhibitor-Resistant SHV β-Lactamases

    PubMed Central

    Winkler, Marisa L.; Papp-Wallace, Krisztina M.; Taracila, Magdalena A.

    2015-01-01

    β-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase inhibitor with activity against many class A and C and some class D β-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of β-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a β-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for blaSHV-1 to 256 mg/liter for blaSHV S130G expressed in Escherichia coli DH10B. At steady state, the k2/K value of the S130G variant when inactivated by avibactam was 1.3 M−1 s−1, versus 60,300 M−1 s−1 for the SHV-1 β-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15–avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the β-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely

  4. Crystallization and X-ray diffraction analysis of an L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and a D-xylonate dehydratase from Caulobacter crescentus.

    PubMed

    Rahman, Mohammad Mubinur; Andberg, Martina; Koivula, Anu; Rouvinen, Juha; Hakulinen, Nina

    2016-08-01

    L-Arabinonate dehydratase (EC 4.2.1.25) and D-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. L-Arabinonate dehydratase converts L-arabinonate into 2-dehydro-3-deoxy-L-arabinonate, and D-xylonate dehydratase catalyzes the dehydration of D-xylonate to 2-dehydro-3-deoxy-D-xylonate. L-Arabinonate and D-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any L-arabinonate or D-xylonate dehydratase is available in the PDB. In this study, recombinant L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT) and D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) were heterologously expressed in Escherichia coli and purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals of RlArDHT that diffracted to 2.40 Å resolution were obtained using sodium formate as a precipitating agent. They belonged to space group P21, with unit-cell parameters a = 106.07, b = 208.61, c = 147.09 Å, β = 90.43°. Eight RlArDHT molecules (two tetramers) in the asymmetric unit give a VM value of 3.2 Å(3) Da(-1) and a solvent content of 62%. Crystals of CcXyDHT that diffracted to 2.66 Å resolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 Å, β = 97.38°. Four CcXyDHT molecules (a tetramer) in the asymmetric unit give a VM value of 4.0 Å(3) Da(-1) and a solvent content of 69%.

  5. Unravelling the Secrets of Mycobacterial Cidality through the Lens of Antisense

    PubMed Central

    Datta, Santanu; Shandil, Radha Krishan; Kumar, Naveen; Robert, Nanduri; Sokhi, Upneet K.; Guptha, Supreeth; Narayanan, Shridhar; Anbarasu, Anand; Ramaiah, Sudha

    2016-01-01

    One of the major impediments in anti-tubercular drug discovery is the lack of a robust grammar that governs the in-vitro to the in-vivo translation of efficacy. Mycobacterium tuberculosis (Mtb) is capable of growing both extracellular as well as intracellular; encountering various hostile conditions like acidic milieu, free radicals, starvation, oxygen deprivation, and immune effector mechanisms. Unique survival strategies of Mtb have prompted researchers to develop in-vitro equivalents to simulate in-vivo physiologies and exploited to find efficacious inhibitors against various phenotypes. Conventionally, the inhibitors are screened on Mtb under the conditions that are unrelated to the in-vivo disease environments. The present study was aimed to (1). Investigate cidality of Mtb targets using a non-chemical inhibitor antisense-RNA (AS-RNA) under in-vivo simulated in-vitro conditions.(2). Confirm the cidality of the targets under in-vivo in experimental tuberculosis. (3). Correlate in-vitro vs. in-vivo cidality data to identify the in-vitro condition that best predicts in-vivo cidality potential of the targets. Using cidality as a metric for efficacy, and AS-RNA as a target-specific inhibitor, we delineated the cidality potential of five target genes under six different physiological conditions (replicating, hypoxia, low pH, nutrient starvation, nitrogen depletion, and nitric oxide).In-vitro cidality confirmed in experimental tuberculosis in BALB/c mice using the AS-RNA allowed us to identify cidal targets in the rank order of rpoB>aroK>ppk>rpoC>ilvB. RpoB was used as the cidality control. In-vitro and in-vivo studies feature aroK (encoding shikimate kinase) as an in-vivo mycobactericidal target suitable for anti-TB drug discovery. In-vitro to in-vivo cidality correlations suggested the low pH (R = 0.9856) in-vitro model as best predictor of in-vivo cidality; however, similar correlation studies in pathologically relevant (Kramnik) mice are warranted. In the acute

  6. Primary biliary cirrhosis is characterized by IgG3 antibodies cross-reactive with the major mitochondrial autoepitope and its Lactobacillus mimic.

    PubMed

    Bogdanos, Dimitrios-Petrou; Baum, Harold; Okamoto, Manabu; Montalto, Paolo; Sharma, Umesh C; Rigopoulou, Eirini I; Vlachogiannakos, John; Ma, Yun; Burroughs, Andrew K; Vergani, Diego

    2005-08-01

    The serological hallmark of primary biliary cirrhosis (PBC) is the presence of pyruvate dehydrogenase complex E2 subunit (PDC-E2) antimitochondrial antibodies (AMAs). Anti-PDC-E2 antibodies cross-react specifically with mycobacterial hsp65, and we have demonstrated that the motif SxGDL[ILV]AE shared by PDC-E2(212-226) and hsp's is a cross-reactive target. Having found that this same motif is present only in beta-galactosidase of Lactobacillus delbrueckii (BGAL LACDE), we hypothesized that this homology would also lead to cross-reactivity. The mimics were tested via ELISA for reactivity and competitive cross-reactivity using sera from 100 AMA-positive and 23 AMA-negative PBC patients and 190 controls. An Escherichia coli (ECOLI) PDC-E2 mimic that has been pathogenetically linked to PBC but lacks this motif has been also tested. Anti-BGAL(266-280) LACDE antibodies were restricted to AMA-positive patients (54 of 95, 57%) and belonged to immunoglobulin (Ig) G3. Of the 190 controls, 22 (12%; P < .001) had anti-BGAL(266-280) antibodies, mainly of the IgG4 subclass. ECOLI PDC-E2 reactivity was virtually absent. BGAL(266-280)/PDC-E2(212-226) reactivity of the IgG3 isotype was found in 52 (52%) AMA-positive PBC patients but in only 1 of the controls (P < .001). LACDE BGAL(266-280)/PDC-E2(212-226) reactivity was due to cross-reactivity as confirmed via competition ELISA. Antibody affinity for BGAL(266-280) was greater than for PDC-E2 mimics. Preincubation of a multireactive serum with BGAL(266-280) reduced the inhibition of enzymatic activity by 40%, while marginal effect (12%) or no effect (2%) was observed in human or ECOLI PDC-E2 mimics. In conclusion, IgG3 antibodies to BGAL LACDE cross-react with the major mitochondrial autoepitope and are characteristic of PBC. PMID:16025495

  7. Characteristics and phylogeny of Bacillus cereus strains isolated from Maari, a traditional West African food condiment.

    PubMed

    Thorsen, Line; Kando, Christine Kere; Sawadogo, Hagrétou; Larsen, Nadja; Diawara, Bréhima; Ouédraogo, Georges Anicet; Hendriksen, Niels Bohse; Jespersen, Lene

    2015-03-01

    Maari is a spontaneously fermented food condiment made from baobab tree seeds in West African countries. This type of product is considered to be safe, being consumed by millions of people on a daily basis. However, due to the spontaneous nature of the fermentation the human pathogen Bacillus cereus occasionally occurs in Maari. This study characterizes succession patterns and pathogenic potential of B. cereus isolated from the raw materials (ash, water from a drilled well (DW) and potash), seed mash throughout fermentation (0-96h), after steam cooking and sun drying (final product) from two production sites of Maari. Aerobic mesophilic bacterial (AMB) counts in raw materials were of 10(5)cfu/ml in DW, and ranged between 6.5×10(3) and 1.2×10(4)cfu/g in potash, 10(9)-10(10)cfu/g in seed mash during fermentation and 10(7) - 10(9) after sun drying. Fifty three out of total 290 AMB isolates were identified as B. cereus sensu lato by use of ITS-PCR and grouped into 3 groups using PCR fingerprinting based on Escherichia coli phage-M13 primer (M13-PCR). As determined by panC gene sequencing, the isolates of B. cereus belonged to PanC types III and IV with potential for high cytotoxicity. Phylogenetic analysis of concatenated sequences of glpF, gmk, ilvD, pta, pur, pycA and tpi revealed that the M13-PCR group 1 isolates were related to B. cereus biovar anthracis CI, while the M13-PCR group 2 isolates were identical to cereulide (emetic toxin) producing B. cereus strains. The M13-PCR group 1 isolates harboured poly-γ-D-glutamic acid capsule biosynthesis genes capA, capB and capC showing 99-100% identity with the environmental B. cereus isolate 03BB108. Presence of cesB of the cereulide synthetase gene cluster was confirmed by PCR in M13-PCR group 2 isolates. The B. cereus harbouring the cap genes were found in potash, DW, cooking water and at 8h fermentation. The "emetic" type B. cereus were present in DW, the seed mash at 48-72h of fermentation and in the final product

  8. Cardiovascular Deconditioning in Humans: Alteration in Cardiovascular Regulation and Function During Simulated Microgravity

    NASA Technical Reports Server (NTRS)

    Cohen, Richard

    1999-01-01

    Alterations in cardiovascular regulation and function that occur during and after space flight have been reported. These alterations are manifested, for example, by reduced orthostatic tolerance upon reentry to the earth's gravity from space. However, the precise physiologic mechanisms responsible for these alterations remain to be fully elucidated. Perhaps, as a result, effective countermeasures have yet to be developed. In this project we apply a powerful, new method - cardiovascular system identification (CSI) - for the study of the effects of space flight on the cardiovascular system so that effective countermeasures can be developed. CSI involves the mathematical analysis of second-to-second fluctuations in non-invasively measured heart rate, arterial blood pressure (ABP), and instantaneous lung volume (ILV - respiratory activity) in order to characterize quantitatively the physiologic mechanisms responsible for the couplings between these signals. Through the characterization of all the physiologic mechanisms coupling these signals, CSI provides a model of the closed-loop cardiovascular regulatory state in an individual subject. The model includes quantitative descriptions of the heart rate baroreflex, autonomic function, as well as other important physiologic mechanisms. We are in the process of incorporating beat-to-beat fluctuations of stroke volume into the CSI technique in order to quantify additional physiologic mechanisms such as those involved in control of peripheral vascular resistance and alterations in cardiac contractility. We apply CSI in conjunction with the two general protocols of the Human Studies Core project. The first protocol involves ground-based, human head down tilt bed rest to simulate microgravity and acute stressors - upright tilt, standing and bicycle exercise - to provide orthostatic and exercise challenges. The second protocol is intended to be the same as the first but with the addition of sleep deprivation to determine whether

  9. Evaluation of a Shuttle Derived Vehicle (SDV) for Cargo Transportation

    NASA Technical Reports Server (NTRS)

    Roman, Jose M.; Meacham, Stephen B.; Krupp, Donald R.; Threet, G. E.; Best, Joel; Davis, Stephan R.; Crumbly, Christopher; Olsen, Ronald A.; Engler, Leah M.; Garner, Tim

    2005-01-01

    In this new era of space exploration, a host of launch vehicles are being examined for possible use in transporting cargo and crew to low Earth orbit and beyond. Launch vehicles derived from the Space Shuttle Program (SSP), known as Shuttle Derived Vehicles (SDVs), are prime candidates for heavy-lift duty because of their potential to minimize non-recurring costs and because the Shuttle can leverage off proven high-performance flight systems with established ground and flight support. To determine the merits of SDVs, a detailed evaluation was performed. This evaluation included a trade study and risk assessment of options based on performance, safety reliability, cost, operations, and evolution. The purpose of this paper is to explain the approach, processes, and tools used to evaluate launch vehicles for heavy lift cargo transportation. The process included defining the trade space, characterizing the concepts, analyzing the systems, and scoring the options. The process also included a review by subject experts from NASA and industry to compare past and recent study data and assess the risks. A set of technical performance measures (TPMs) was generated based on the study requirements and constraints. Tools such as INTROS and POST were used to calculate performance, FIRST was used for prediction of reliability, and other software packages, both commercial and NASA-owned, were applied to study the trade space. By following a clear process and using the right tools a thorough assessment was performed. An SDV can be classified as either a side-mount vehicle (SMV) or an in-line vehicle OLV). An SMV is a Space Shuttle where the Orbiter is replaced by a cargo carrier. An ILV is comprised of a modified Shuttle External Tank (ET) with engines mounted to the bottom and cargo mounted atop. For both families of vehicles, Solid Rocket Boosters (SRBs) are attached to the ET. The first derivate of Shuttle is defined as the vehicle with minimum changes necessary to transform the

  10. EDITORIAL: Focus on Mechanical Systems at the Quantum Limit FOCUS ON MECHANICAL SYSTEMS AT THE QUANTUM LIMIT

    NASA Astrophysics Data System (ADS)

    Aspelmeyer, Markus; Schwab, Keith

    2008-09-01

    progress was reported almost on a monthly basis and new groups entered the field. We intend to keep submission to this Focus Issue open for some time and invite everyone to share their latest results with us. And finally, a note to our fellow colleagues: keep up the good work! We would like to call the next Focus Issue 'Mechanical Systems IN the Quantum Regime'. Focus on Mechanical Systems at the Quantum Limit Contents Parametric coupling between macroscopic quantum resonators L Tian, M S Allman and R W Simmonds Quantum noise in a nanomechanical Duffing resonator E Babourina-Brooks, A Doherty and G J Milburn Creating and verifying a quantum superposition in a micro-optomechanical system Dustin Kleckner, Igor Pikovski, Evan Jeffrey, Luuk Ament, Eric Eliel, Jeroen van den Brink and Dirk Bouwmeester Ground-state cooling of a nanomechanical resonator via a Cooper-pair box qubit Konstanze Jaehne, Klemens Hammerer and Margareta Wallquist Dissipation in circuit quantum electrodynamics: lasing and cooling of a low-frequency oscillator Julian Hauss, Arkady Fedorov, Stephan André, Valentina Brosco, Carsten Hutter, Robin Kothari, Sunil Yeshwanth, Alexander Shnirman and Gerd Schön Route to ponderomotive entanglement of light via optically trapped mirrors Christopher Wipf, Thomas Corbitt, Yanbei Chen and Nergis Mavalvala Nanomechanical-resonator-assisted induced transparency in a Cooper-pair box system Xiao-Zhong Yuan, Hsi-Sheng Goan, Chien-Hung Lin, Ka-Di Zhu and Yi-Wen Jiang High-sensitivity monitoring of micromechanical vibration using optical whispering gallery mode resonators A Schliesser, G Anetsberger, R Rivière, O Arcizet and T J Kippenberg Optomechanical to mechanical entanglement transformation Giovanni Vacanti, Mauro Paternostro, G Massimo Palma and Vlatko Vedral The optomechanical instability in the quantum regime Max Ludwig, Björn Kubala and Florian Marquardt Quantum limits of photothermal and radiation pressure cooling of a movable mirror M Pinard and A Dantan

  11. Engineering Ralstonia eutropha for Production of Isobutanol (IBT) Motor Fuel from Carbon Dioxide, Hydrogen, and Oxygen Project Final Report

    SciTech Connect

    Sinskey, Anthony J.; Worden, Robert Mark; Brigham, Christopher; Lu, Jingnan; Quimby, John Westlake; Gai, Claudia; Speth, Daan; Elliott, Sean; Fei, John Qiang; Bernardi, Amanda; Li, Sophia; Grunwald, Stephan; Grousseau, Estelle; Maiti, Soumen; Liu, Chole

    2013-12-16

    into complex cellular molecules using the energy from hydrogen. In this research project, engineered strains of R. eutropha redirected the excess carbon from PHB storage into the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can be used directly as substitutes for fossil-based fuels and are seen as alternative biofuels to ethanol and biodiesel. Importantly, these alcohols have approximately 98 % of the energy content of gasoline, 17 % higher than the current gasoline additive ethanol, without impacting corn market production for feed or food. Unlike ethanol, these branched-chain alcohols have low vapor pressure, hygroscopicity, and water solubility, which make them readily compatible with the existing pipelines, gasoline pumps, and engines in our transportation infrastructure. While the use of alternative energies from solar, wind, geothermal, and hydroelectric has spread for stationary power applications, these energy sources cannot be effectively or efficiently employed in current or future transportation systems. With the ongoing concerns of fossil fuel availability and price stability over the long term, alternative biofuels like branched-chain higher alcohols hold promise as a suitable transportation fuel in the future. We showed in our research that various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, would produce isobutanol and 3-methyl-1-butanol when initiated during nitrogen or phosphorus limitation. Early on, we isolated one mutant R. eutropha strain which produced over 180 mg/L branched-chain alcohols in flask culture while being more tolerant of isobutanol toxicity. After the targeted elimination of genes encoding several potential carbon sinks (ilvE, bkdAB, and ace