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     

Screening costramid libraries for chromosomal genes: an alternative interspecific hybridization method

Abstract Broad host-range RK2-based cosmid vectors ('costramids') are increasingly used in molecular genetic studies of Gram-negative soil bacteria such as Rhizobium spp. we describe a simple modification of existing methods, whereby a genomic library constructed in a stringently replicated vector can be screened for genes which are undetectable by colony hybridization due to background cross-hybridization. This method allows the use of 'heterologous' probes (interspecies hybridization) to isolate several presumptive genes of interest from a gene bank of Rhizobium sp. NGR234 made in the costramid pRK7813. These are a gene with homology to the citrate synthase gene ( gltA ) or Escherichia coli , the gene encoding δ-aminol evulinic acid synthase ( hemA ), and a gene or genes regulating dicarboxylate transport.     

Dietary unsaturated fatty acids: interactions and possible needs in relation to eicosanoid synthesis

In addition to providing energy and essential fatty acids, dietary fatty acids can affect numerous biochemical and physiologic reactions related to secretory, cardiovascular, and immune functions. The major dietary unsaturated fatty acid, linoleic acid, affects tissue arachidonic acid and can influence eicosanoid-mediated reactions. Chronic, excess, or imbalanced eicosanoid synthesis may be conductive to excessive inflammation, thrombotic tendencies, atherosclerosis, and immune suppression. Dietary n-3 polyunsaturated fatty acids (PUFAs) may ameliorate eicosanoid-related phenomena by reducing tissue arachidonic acid and by inhibiting eicosanoid synthesis. This review summarizes information concerning the metabolism of unsaturated fatty acids, with emphasis on tissue arachidonic acid levels and eicosanoids, and discusses the need for data concerning the appropriate intake of dietary n-6 and n-3 PUFAs to modulate arachidonic acid and eicosanoid synthesis and to minimize possible adverse reactions.     

Cloning of hemA from Rhizobium sp. NGR234 and symbiotic phenotype of a gene-directed mutant in diverse legume genera

Summary The hemA gene which encodes -aminolaevulinic acid synthase (ALAS), was cloned and characterized from the broad host-range Rhizobium strain NGR234. A cosmid, identified by hybridization with the cloned gene of R. meliloti and complementation of an R. meliloti hemA mutant, was subcloned to yield a 5.5 kb fragment containing the entire NGR234 gene. A physical-genetic map was made and the interposon was introduced into a single EcoRI site which bisects the gene. The mutated gene was homogenotized into NGR234 to generate a hemA mutant, with a view to evaluating the role of rhizobial bacteroid ALAS activity for a wide variety of legume symbioses. The mutant strain formed an ineffective (Fix^–) symbiosis with all tested host plants. These included tropical legumes that produce either indeterminate (Leucaena) or determinate (Desmodium, Macroptilium, Lablab, Vigna) root nodules.Abbreviations ALA -aminolaevulinic acid - ALAS aminolaevulinic acid synthase - Lb leghaemoglobin - Lb-haem haem moiety of leghaemoglobin     

Molecular and Cellular Analysis of the DNA Repair Defect in a Patient in Xeroderma Pigmentosum Complementation Group D Who Has the Clinical Features of Xeroderma Pigmentosum and Cockayne Syndrome

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are quite distinct genetic disorders that are associated with defects in excision repair of UV-induced DNA damage. A few patients have been described previously with the clinical features of both disorders. In this paper we describe an individual in this category who has unusual cellular responses to UV light. We show that his cultured fibroblasts and lymphocytes are extremely sensitive to irradiation with UV-C, despite a level of nucleotide excision repair that is 30%–40% that of normal cells. The deficiency is assigned to the XP-D complementation group, and we have identified two causative mutations in the XPD gene: a gly→arg change at amino acid 675 in the allele inherited from the patient's mother and a −1 frameshift at amino acid 669 in the allele inherited from his father. These mutations are in the C-terminal 20% of the 760-amino-acid XPD protein, in a region where we have recently identified several mutations in patients with trichothiodystrophy.     

Synthesis and gastrointestinal pharmacology of some 15- and 16- modified (±)-11-deoxyprostaglandins

The synthesis and gastrointestinal pharmacology of some 11-deoxyprostaglandin E₁ analogues are described with results analysed for selectivity from side effects. 11-Deoxygenation reduced potency relative to PGE₂ but, as has been reported for natural PGs, 15- or 16-methyl analogues were more potent than the unsubstituted parent compound in the order 16-methyl > 15-methyl > 16, 16-dimethyl. The results suggest that a complex interaction between C-15 and C-16 in methyl analogues affects their profile of activity, but that none of the modifications studied conferred a substantial potency or selectivity advantage over PGE₂.     

Isolation and metabolism of Vigna unguiculata root nodule protoplasts

Axenic cultures of bacteroid-containing protoplasts were isolated from root nodules of Vigna unguiculata L. Walp. Dimensions of the protoplasts were 35 to 135 m long x 35 to 95 m wide. Yields were about 30 to 50 mg dry weight per gram fresh weight of nodules. About 5x10⁸ protoplasts packed into 1 ml of basal medium under the influence of gravity. When incubated in hypertonic, nitrogen-free media, freshly isolated protoplasts began to reduce acetylene to ethylene after a lag period of 24 to 48 h. Various additions to the basal medium showed that the system possessed functional glycolytic and tricarboxylic acid pathways. Endogenous application of various intermediary metabolites stimulated both acetylene reduction and respiration, though not often equally. As acetylene reduction, but not respiration, was inhibitable by both asparagine and glutamine, the system appears suitable for the study of mechanisms controlling symbiotic nitrogen fixation.Abbreviations BSA bovine serum albumine - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PEP phospho(enol)pyruvate - UMKL 76 University of Malaga, Kuala Lumpur, Rhizobium, No. 76 - TCAC tricarboxylic acid cycle     

The effect of prostaglandin E2 on the cardiovascular response to angiotensin II in pregnant rabbits

The rise in arterial blood pressure in response to angiotensin II was studied in the last third of pregnancy in rabbits. The response was compared with that of pregnant rabbits during infusion of prostaglandin E₂ and F_2α. Prostaglandin E₂ significantly diminished the rise in diastolic pressure in response to angiotensin II. Prostaglandin F_2α did not alter the response. Intravenous indomethacin elevated the blood pressure and caused an absolute increase in the pressor response. It did not mediate a change in the percentage rise in blood pressure in response to angiotensin II.     

Carotenoid pigments in a red strain of Rhizobium from Lotononis bainesii Baker

The carotenoid pigments of a Rhizobium strain isolated from Lotononis bainesii were found to be diglucosyl-4,4-diapocarotene-4,4-dioate and glucosyl-4,4-diapocarotene-4-oate-4-oic acid.5th publication in the series Carotenoids of Rhizobia [4th publication: Helv. chim. Acta 62: 2551–2557 (1979)]