Improved L-lysine production by the amplification of theCorynebacterium glutamicum dapA gene encoding dihydrodipicolinate synthetase inE. coli

Summary ThedapA gene (L-2,3-dihydrodipicolinate synthetase: DHDP synthetase) ofCorynebacterium glutamicum JS231, a lysine overproducer, cloned and subcloned inE. coli/C. glutamicum shuttle vector pECCG117 was used to transformE. coli threonine producer and threonine and lysine coproducer. The plasmid pDHDP5812 carryingdapA gene ofC. glutamicum led to increase in lysine production in theseE. coli strains. Threonine and lysine co-producerE. coli TF1 with pDHDP5812 produced lysine with small amount of threonine. The DHDP synthetase activity ofE. coli TF1 carrying pDHDP5812 showed high resistance toward inhibition by lysine.     

Non-polluting wood and pulp delignification: Biomimetic ligninase system

Summary We report the delignification ofPinus radiata D Don,Eucalyptus globulus andEucalyptus grandis woods (formic acid treated and untreated) by 2 h treatment with a hemin/hydrogen peroxide system. The untreated chips and sawdust ofE. globulus were 30% and 50% delignified respectively. No significant effects were found forP. radiata sawdust;P. radiata treated chips (organosolv pulp) did not show any further delignification upon hemin/peroxide action, 25% delignification was achieved in untreated chips. In the case ofE. grandis untreated wood the delignification was better in sawdust than in chips, but in smaller percentage than in the otherEucalyptus species. This relation is maintained in substrates, treated with formic acid or untreated. The delignification of chips in both species ofEucalyptus was improved when they were pre-treated with formic acid. The loss of lignin in theE. grandis andE. globulus sawdust (pre-treated with formic acid) was 79% and 75% respectively.     

Endocytic uptake of Escherichia coli spheroplasts by Neurospora crassa slime cells

Summary Interaction of Escherichia coli spheroplasts with Neurospora crassa slime cells was examined by transmission electron microscopy after treatment with polyvinyl alcohol followed by dilution with the high pH-high Ca buffer. Bacterial spheroplasts were found either adhering to the flat surface, associating with the invaginating surface, or residing within the intracellular vesicle of fungal protoplasts. In addition, bacterial spheroplasts free of the surrounding vesicles and those in the course of breakdown were observed in the fungal cytoplasm. It was concluded that Escherichia coli spheroplasts are taken up by Neurospora crassa protoplasts almost exclusively via endocytosis. This is the first cytological evidence for the endocytic activity of fungal cells.     

Building Better Barrels – β-barrel Biogenesis and Insertion in Bacteria and Mitochondria

β-barrel proteins are folded and inserted into outer membranes by multi-subunit protein complexes that are conserved across different types of outer membranes. In Gram-negative bacteria this complex is the barrel-assembly machinery (BAM), in mitochondria it is the sorting and assembly machinery (SAM) complex, and in chloroplasts it is the outer envelope protein Oep80. Mitochondrial β-barrel precursor proteins are translocated from the cytoplasm to the intermembrane space by the translocase of the outer membrane (TOM) complex, and stabilized by molecular chaperones before interaction with the assembly machinery. Outer membrane bacterial BamA interacts with four periplasmic accessory proteins, whereas mitochondrial Sam50 interacts with two cytoplasmic accessory proteins. Despite these major architectural differences between BAM and SAM complexes, their core proteins, BamA and Sam50, seem to function the same way. Based on the new SAM complex structures, we propose that the mitochondrial β-barrel folding mechanism follows the budding model with barrel-switching aiding in the release of new barrels. We also built a new molecular model for Tom22 interacting with Sam37 to identify regions that could mediate TOM-SAM supercomplex formation.     

Bifunctional cosmid vector forStreptomyces

Summary AnEscherichia coli/Streptomyces bifunctional cosmid vector, pJP3, which selects for clones with insert size 23–33 kilobase pairs (kb), has been constructed. A genomic DNA library fromS. kanamyceticus has been stably maintained inE. coli using pJP3. This recombinant DNA can be transferred intoS. lividans with increased efficiency by utilizing heat treatment during protoplast formation.     

Transformation ofBacillus subtilis with the treatment by alkali cations

Summary ExposingBacillus subtilis cultures to high concentrations of alkali cations, especially K⁺, allows efficient transformation by plasmids. The method allows transformation with unfractionated plasmid DNA, monomeric plasmid DNA as well as linear plasmid DNA.B. subtilis strains, not amenable to natural transformation, were also transformed by the present method.     

Source of pyrrole-2-carboxylate in mammalian urine

Pyrrole-2-car?ylate, earlier reported in human urine and labeled in rat urine after administration of radioactive proline, arises more directly from labeled hydroxyproline. Antibiotic treatment appeared to exclude epimerization of administered hydroxy-L-proline to a D-epimer by intestinal bacteria. A likely reaction for the in vivo conversion is hydroxy-L-proline oxidation by the L-amino acid oxidase of rat kidney, demonstrable with purified enzyme. Crystalline D-amino acid oxidase also catalyzes a slow oxidation of hydroxy-L-proline. These two reactions are adequate to account for the normal excretion of pyrrole-2-car?ylate by a number of species.     

Regeneration of plants from callus tissue of the pasture legume Centrosema brasilianum

Plants were regenerated from leaf explants of Centrosema brasilianum cultured in vitro. Callus and buds were produced on Murashige and Skoog medium (MS), 0.8% agar, 0.1 mg/l NAA and 1 mg/l BAP. Regeneration of multiple shoots was achieved by transferring callus onto fresh medium containing 0.01 and 1 mg/l of NAA and BAP, respectively. Shoots formed roots upon transfer to MS with 0.01 mg/l NAA. Plantlets were succesfully transferred to soil. Leaf-derived calli of Centrosema arenarium, C. macrocarpum, C. pascuorum, C. pubescens, and C. virginianum did not produce shoots when cultured in vitro.     

Structural and SAXS analysis of the budding yeast SHU-complex proteins

In Saccharomyces cerevisiae, four proteins, Shu1, Shu2, Psy3 and Csm2, form a stable SHU-complex both in vivo and in vitro. These proteins are involved in the early stages of the homologous recombination DNA damage repair process. In this paper, the crystal structure of the Psy3–Csm2 sub-complex is presented at 1.8 Å resolution and successfully fitted into our small angle X-ray scattering (SAXS) data of the SHU-complex. Taken together with our electrophoretic mobility shift assay (EMSA) results, a model is proposed for the SHU–protein complex coupled with DNA.Structured summary of protein interactions:PSY3 and CSM2 bind by X-ray crystallography (View interaction) PSY3, CSM2, Shu 1 and Shu 2 physically interact by x ray scattering (View interaction)     

<Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> type III secretion: Evidence for the ability to transport proteins that are folded prior to secretion

Background  

Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis) share a type three secretion system (TTSS) which allows translocation of effector proteins (called Yops) into host cells. It is believed that proteins are delivered through a hollow needle with an inner diameter of 2–3 nm. Thus transport seems to require substrates which are essentially unfolded. Recent work from different groups suggests that the Yersinia TTSS cannot accommodate substrates which are folded prior to secretion. It was suggested that folding is prevented either by co-translational secretion or by the assistance of specific Yop chaperones (called Sycs).     

Expression ofBacillus subtilis endoglucanase gene inLactobacillus acidophilus

Summary ABacillus subtilis gene which codes for endoglucanase was transferred intoLactobacillus acidophilus by electroporation with plasmids containing the gene. The endoglucanase gene expressed well in the transformed cells and most of the gene product was found in the culture medium. The efficiency of the endoglucanase production by the transformed.L. acidophilus cells depended greatly on the choice of the vector plasmids on which the gene to be inserted.     

Production of tropane alkaloids in cultured cells of Hyoscyamus niger

Tests for calluses rich in tropane alkaloids were made with newly induced calluses of Atropa belladonna, Datura stramonium and Hyoscyamus niger. Only calluses of H. niger gave an alkaloid-positive test.A Hyoscyamus cell line had the highest total alkaloid content of all the calluses screened by the cell-squash alkaloid assay. Both hyoscyamine and scopolamine were identified in the cultured cells of this line by TLC, GLC and GC-MS.Abbreviations NAA 1-Naphthaleneacetic acid - BA Benzyladenine - BSA N,O-Bis(trimethylsilyl) acetamide     

pIDDLE6: A system for ligation and expression-cloning in E. coli

Summary The pIDDLE6 (for PCR-based, in-frame, directional DNA ligation and expression) system utilises novel ligation procedures to clone and overexpress almost any coding sequence. Inserts are cloned either by blunt ligation in the presence of PmeI or by ligation between dissimilar SfiI sites. A chimeric protein with both N- and C-terminal tags is produced, allowing purification by via starch-or nickel-affinity under native or denaturing conditions. The tags can be removed independently. The the vector and ligation procedures have been used successfully as described.     

Species specific shoot regeneration response of cotyledonary explants of Brassicas

A study of shoot regeneration from cotyledons of three basic diploid species of Brassica, B. campestris (AA), B. nigra (BB), B. oleracea (CC) and their amphidiploids B. juncea (AABB), B. napus (AACC) and B. carinata (BBCC) showed species-specific responses for in vitro shoot regeneration. Analysis of the species mean shoot regeneration response over a range of growth regulator combinations revealed that i) B. campestris is the lowest regenerating species, ii) B. nigra and B. oleracea regenerate with high frequencies, iii) In amphidiploids, the presence of B. campestris component brings down shoot regeneration frequency below the value of B. oleracea in B. napus combination and is additive of the combining genomes in B. juncea combination. In B. carinata regeneration frequencies are less than the parental diploid species, iv) Significant intraspecific genotypic differences were observed for B. nigra and B. oleracea among diploids and B. juncea and B. carinata among amphidiploids, when cotyledons of eighteen genotypes were tested in one growth regulator combination.Abbreviations MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - IAA Indole 3-acetic acid - BA 6-Benzyl aminopurine     

A novel method to produce Anabaena-free Azolla by in vitro fertilization of micromanipulated megasporocarps

In the Azolla-Anabaena azollae symbiotic system, Anabaena akinetes get entrapped between the indusium and the apical cap of the megaspore apparatus during megasporocarp development, thus maintaining the continuity of the cyanobacterial association throughout the life cycle of the fern. The entrapped akinetes serve as the source of inoculum for infecting the new sporophyte when it is emerging from the megaspore apparatus. A procedure to generate Anabaena-free Azolla was developed by fertilizing the germinating megasporocarps in which the indusium along with the akinetes were removed by micromanipulation. This method has the advantage of not requiring drastic treatments of Azolla with antibiotics to eliminate the endosymbiotic cyanobacterial cells. Details of this new method and its usefulness in studies aimed at recombination of Azolla with Anabaena azollae are discussed.Abbreviations IRRI International Rice Research Institute - I^– IRRI medium devoid of combined nitrogen - I⁺ IRRI medium containing combined nitrogen - SDS sodium dodecyl sulfate     

The effect of streptomycin and hypotonicity on survival of Pseudomonas aeruginosa

Summary P. aeruginosa proliferates well in a water environment; however, when subjected to high doses of streptomycin or gentamicin, the residual viable bacteria are killed by moderate water dilution of their media. These results lead to the suggestion that the mechanism of lethal action of aminoglycosides may operate through interference with the water balance system of the P. aeruginosa.     

Nucleosides,I Ribosylation of 8-Substituted Theophylline Derivatives

Abstract

The attempted ribosylation reaction of 8-nitro-theophylline (2) with 1-o-acetyl-2, 3, 5-tri-o-benzoyl-D-ribo-furanose (5) failed to give any nucleoside product, whereas the reaction of 8-chlorotheophylline (3) with 5 afforded the 8-chloro-7-(2,3,5-tri-o-benzoyl) β-D-ribofuranosyltheophylline (6) in good yield. The product 6 reacted with benzylamine producing the 8-benzylamino-7-(2, 3, 5-tri-O-benzoyl) β-D-ribo-furanosyltheophylline (10), which could also be synthesised by ribosylation of 8-benzylaminotheophylline (8) with 5. Debenzoylation of 6 and 10 gave the corresponding 7-β-D-ribofuranosyltheophylline nucleosides (7) and (11), respectively. Compound 7 could be converted into 11 by reaction with benzylamine. The newly synthesised compounds have been characterised by elemental analysis, ¹H-NMR and UV spectra.     

Thermostable tryptophan synthase ofBacillus stearothermophilus expressed inEscherichia coli

Summary The tryptophan synthase genes,trpA andtrpB, from a moderate thermophile,Bacillus stearothermophilus IFO13737, were expressed efficiently inEscherichia coli. The recombinant tryptophan synthase amounted to 22% of the soluble cellular protein, and was purified to homogeneity by three steps. The enzyme is more thermostable thanE.coli tryptophan synthase, especially the subunit. The enzyme is also more resistant to sodium dodecylsulfate and methanol thanE.coli enzyme.     

Comparative shoot regeneration responses of diploid brassicas and their synthetic amphidiploid products

Comparative shoot regeneration responses of three diploid Brassica species, B. campestris (AA), B. nigra (BB) and B. oleracea (CC) and their synthetic amphidiploid combinations have been investigated. The study indicates that A genome has an inhibitory effect on regeneration as evident from significantly low responses of B. juncea (AABB) and B. napus (AACC) combinations, in comparison with regeneration response of B. nigra and B. oleracea. This inhibition may arise from genomic interactions or from the B. campestris cytoplasm interacting negatively with the alien genome. Significant cytoplasmic influence on regenerability has been observed in B. carinata (BBCC) synthesised from reciprocal crosses of B. nigra and B. oleracea.Abbreviations MS Murashige and Skoog (1962) - IAA Indole-3-acetic acid - NAA -napthaleneacetic acid - BA 6-Benzylaminopurine     

Nucleosides. 131. The Synthesis of 5-(2-Chloro-2-Deoxy-β-D-arabino-Furanosyl)Uracil. Reinterpretation of Reaction of ψ-Uridine With α-Acetoxyisobutyryl Chloride. Studies Directed Toward the Synthesis of 2′-Deoxy-2′-Substituted Arabino-Nucleosides. 2

Abstract

Treatment of ψ-uridine (3) with α-acetoxyisobutyryl chloride in acetonitrile gave, after deprotection, a mixture of four products: 5-(2-chloro-2-deoxy-β-D-arabinofuranosyl)uracil (10a), its 3′-chloro xylo isomer (11a), 2′-chloro-2′-deoxy-ψ-uridine (9a) and 4,2′-anhydro-ψ-uridine (8a). Each component was isolated by column chromatography. Compound 9 was converted to the known 1,3-dimethyl derivative 2 by treatment with DMF-dimethylacetal. Treatment of 10 and 11 with NaOMe/MeOH afforded the same 4,2′-anhydro-C-nucleoside 8. The 1,3-dimethyl analogues of 10 and 11, however, were converted to 2′,3′-anhydro-1,3-dimethyl-ψ-uridine (13) upon base treatment. The epoxide 13 was also prepared in good yield by treatment of 10 and 11 with DMF-dimethylacetal.