Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis — the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly. Journal of Industrial Microbiology & Biotechnology (2001) 27, 399–402. Received 31 May 2001/ Accepted in revised form 09 July 2001     

Cyclo(His-Pro) up-regulates heme oxygenase 1 via activation of Nrf2-ARE signalling

Paraquat (1,1'-dimethyl-4,4'-bipyridinium), a widely used non-selective herbicide, is a redox cycling agent with adverse effects on dopamine systems. Epidemiological data have shown that exposure to paraquat is one of the several risk factors for Parkinson's disease. We have already shown that cyclo(His-Pro), an endogenous cyclic dipeptide produced by the cleavage of the thyrotropin releasing hormone, has a cytoprotective effect through a mechanism involving Nrf2 activation that decreases production of reactive oxygen species and increases glutathione synthesis. Using primary neuronal cultures and PC12 cells as targets of paraquat neurotoxicity, we addressed whether and how cyclo(His-Pro) causes cellular protective response against paraquat-mediated cell death. We found that cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion by up-regulating Nrf2 gene expression, triggering its nuclear accumulation and activating the expression of heme oxygenase1. These protective effects were abolished by RNA interference-mediated Nrf2 knock down whereas were unaffected by RNA interference-mediated Keap1 knock down. Inhibition of heme oxygenase activity decreased cyclo(His-Pro)-induced neuroprotection. These results suggest that cyclo(His-Pro), acting as a selective activator of the brain modulable Nrf2 pathway, may be a promising candidate as neuroprotective agent that act through induction of phase II genes.     

The nitrite reductase gene of Pseudomonas aeruginosa: Effect of growth conditions on the expression and construction of a mutant by gene disruption

Abstract The expression of nitrite reductase has been tested in a wild-type strain of Pseudomonas aeruginosa (Pao1) as a function of nitrate concentration under anaerobic and aerobic conditions. Very low levels of basal expression are shown under non-denitrifying conditions (i.e. absence of nitrate, in both aerobic and anaerobic conditions); anaerobiosis is not required for high levels of enzyme production in the presence of nitrate. A Pseudomonas aeruginosa strain, mutated in the nitrite reductase gene, has been obtained by gene replacement. This mutant, the first of this species described up to now, is unable to grow under anaerobic conditions in the presence of nitrate. The anaerobic growth can be restored by complementation with the wild-type gene.     

Left ventricular hypertrophy: physiopathological signs using a hemodynamic and cardio-autonomic approach]

The presence of left ventricular hypertrophy (LVH) in either hypertensives -H- or in normotensives -N-, suggests that not only blood pressure is determining this anatomic change, but various factors, as neural or endocrine ones, could be involved in its genesis. In order to evaluate the role of sympathetic dys-reactivity on LVH, we studied three groups of subjects: a) 12 -H- (SBP 159+/-9; DBP 99.6+/-7; FC 80+/-7) with LVH, diagnosed by echocardiogram. b) 12 -N- (SBP 138.2+/-8; DBP 83+/-2; FC 75.6+/-4) with LVH. c) 12 -N- (SBP 136.6+/-11; DBP 81.8+/-5; FC 76.3+/-5) without LVH. Using computer interfaced equipment, we measured beat to beat, hemodynamic and extra-cardiovascular autonomic functions, during a session of stressors (Mental Arithmetic, Color Word Stroop, Cold Pressure and Handgrip Tests), preceded and followed by 10' of observation. Among the various considered indexes, we evaluated the Percentual Total Activity Index (PTAI), as percentual total activity change + percentual total recovery change. Our findings point out that the PTAI of N with LVH is significantly higher for SCL, PHT, HR, SV, CO, TPR than either in H with LVH or N without LVH. These data seem to demonstrate a prolonged reactivity in N without LVH and are according to the hypothesis that LVH could also be supported by a hyper-adrenergic state with sympathetic dys-reactivity, independently from high blood pressure values.     

Persistence of introduced Bradyrhizobium japonicum strains in forming nodules in subsequent years after inoculation in Wisconsin soils

Nodulation of soybeans by indigenous and inoculum strains of Bradyrhizobium japonicum was studied in field experiments in Wisconsin from 1983 to 86. Aqueous suspensions of bacteria were applied to seeds at the time of planting at levels of 7?×?10(7)-10(10) bacteria per 2.5-cm row. The predominant indigenous serogroup was 123 in these soils. Six different inoculum strains were used (two from serocluster 123, two from serogroup 110, and one each from serogroups 122 and C1). Nodule occupants were identified using spontaneous antibiotic-resistant mutations in the inoculum strains, phage typing, and serotyping. In the 1983 experiment, the majority of nodules were formed by the inoculum strains in almost all cases (up to 100% in some cases), in two different soils containing 3.5?×?10(5) indigenous B. japonicum per gram. After 2 years without inoculation at the same two site, the inoculum strains did not form many nodules on uninoculated soybeans (less than 10% in most cases; less than 30% in all cases). In inoculation experiments carried out in 1985 and 1986, four inoculum strains were used (3 members of 123 serocluster and USDA 110str); inocula containing 10(8) bacteria per 2.5-cm row formed less than42%ofthe nodules in soils containing 1?×?10(4)-4?×?10(4)B. japonicum per gram. The major conclusions are (i) the success of inoculation in Midwestern U.S. soils is highly variable, even with members of the (highly competitive) 123 serocluster, and (ii) successful inoculation in 1 year in a Wisconsin soil does not ensure that the inoculated strain will persist in forming nodules in that field in subsequent years without further inoculation. Key words: Bradyrhizobium japonicum, strain persistence, field trials.     

Attempts to Detect Agrobacterium tumefaciens DNA in Crown-Gall Tumor Tissue

Primary and secondary crown gall tissue cultures were established from sunflower plants (Helianthus annuus, variety Mammoth Russian) wound-inoculated with Agrobacterium tumefaciens (Smith and Townsend) Conn strain B(6). Growth rates of tumor tissues and habituated healthy sunflower stem section tissues on basal medium lacking auxin and cytokinin were compared to those of healthy sunflower stem section tissue grown on the same medium with added phytohormones. No difference was detected in the thermal denaturation midpoints (74.8 C) and melting profiles in 25 mm sodium phosphate (pH 6.8), or the buoyant densities in cesium chloride equilibrium centrifugation (1.687 g cm(-3)), between deoxyribonucleic acids (DNAs) isolated from crude nuclear preparations of the four tissue types. No satellite DNA was observed in equilibrium centrifugation of unsheared plant DNAs.Heterologous DNA renaturation kinetic analyses were performed in 0.14 m sodium phosphate (pH 6.8) at 70 C. Thermal stability measurements of reassociated DNA revealed less than 1% of mismatched base pairs. Reannealing of sheared, denatured, radioactive A. tumefaciens B(6) DNA (molecular weight, 325,000 daltons) in the presence of a 5400-fold excess of sheared calf thymus, healthy tissue, or secondary sunflower crown gall DNA obeyed second order kinetics, with a Cot((1/2)) of 2.8, identical to that observed when B(6) DNA was reannealed in the absence of foreign DNA.Reannealing rates of B(6) DNA in the presence of 5400-fold excesses of DNA from two lines of primary sunflower crown gall were increased 2.24- or 1.47-fold. Digestion of the tumor DNA preparations with pancreatic deoxyribonuclease I until no detectable DNA remained, followed by restoration of solution viscosity by added calf thymus DNA, failed to remove the acceleration effect of the tumor DNA preparations. Reisolation of the reannealed nucleic acid formed in this experiment, and digestion with ribonuclease A or deoxyribonuclease I revealed that the double-stranded fraction was composed entirely of DNA-DNA duplexes, with no detectable DNA-RNA hybrids.The data indicate that tumor, but not healthy tissue DNA preparations contain some factor or factors (not DNA) which accelerate the reannealing of bacterial DNA. Sunflower tumor tissue DNAs, therefore, do not contain integrated A. tumefaciens DNA sequences in amounts greater than a random (1/5) of the bacterial genome per diploid amount of plant DNA, or a complete bacterial genome per five diploid plant cell DNA equivalents. Further, the possibility of the presence of many copies of a specific portion greater than 5% of the bacterial genome is excluded.     

Uniparental isodisomy of chromosome 2 causing MRPL44-related multisystem mitochondrial disease

Mutations in nuclear-encoded protein subunits of the mitochondrial ribosome are an increasingly recognised cause of oxidative phosphorylation system (OXPHOS) disorders. Among them, mutations in the MRPL44 gene, encoding a structural protein of the large subunit of the mitochondrial ribosome, have been identified in four patients with OXPHOS defects and early-onset hypertrophic cardiomyopathy with or without additional clinical features. A 23-year-old individual with cardiac and skeletal myopathy, neurological involvement, and combined deficiency of OXPHOS complexes in skeletal muscle was clinically and genetically investigated. Analysis of whole-exome sequencing data revealed a homozygous mutation in MRPL44 (c.467 T?>?G), which was not present in the biological father, and a region of homozygosity involving most of chromosome 2, raising the possibility of uniparental disomy. Short-tandem repeat and genome-wide SNP microarray analyses of the family trio confirmed complete maternal uniparental isodisomy of chromosome 2. Mitochondrial ribosome assembly and mitochondrial translation were assessed in patient derived-fibroblasts. These studies confirmed that c.467 T?>?G affects the stability or assembly of the large subunit of the mitochondrial ribosome, leading to impaired mitochondrial protein synthesis and decreased levels of multiple OXPHOS components. This study provides evidence of complete maternal uniparental isodisomy of chromosome 2 in a patient with MRPL44-related disease, and confirms that MRLP44 mutations cause a mitochondrial translation defect that may present as a multisystem disorder with neurological involvement.

     

Benthic marine flora in the Tuscan Archipelago. A first contribution: Isles of Capraia,Elba, Formiche di Grosseto,Giglio, Scoglio d'Africa,Montecristo and Giannutri

Abstract

Benthic marine flora of Tuscan Archipelago has been studied. Several samples were collected along five islands and some rocks. This paper is a compendium of new and already published data, in which 267 species, varieties, forms and stadia of algae and seagrasses are listed: 5 Bangiophyceae, 182 Florideophyceae, 45 Phaeophyceae, 6 Chlorophyceae, 28 Bryopsidophyceae, 1 Angiospermae.