A highly inflammatory and vesicatory substance, lyngbyatoxin A, has been isolated from the lipid extract of a Hawaiian shallow-water variety of Lyngbya majuscula Gomont; its gross structure was determined from chemical and spectral data. Lyngbyatoxin A is closely related to teleocidin B, a poisonous substance associated with several strains of Streptomyces.

Two isolates of Paenibacillus validus (DSM ID617 and ID618) stimulated growth of the arbuscular mycorrhizal fungus Glomus intraradices Sy167 up to the formation of fertile spores, which recolonize carrot roots. Thus, the fungus was capable of completing its life cycle in the absence of plant roots, but relied instead on the simultaneous growth of bacteria. The supernatant of a mixed batch culture of the two P. validus isolates contained raffinose and another, unidentified trisaccharide. Among the oligosaccharides tested, raffinose was most effective in stimulating hyphal mass formation on plates but could not promote growth to produce fertile spores. A suppressive subtractive hybridization library followed by reverse Northern analyses indicated that several genes with products involved in signal transduction are differentially expressed in G. intraradices SY 167 when grown in coculture with P. validus (DSM 3037). The present investigation, while likely representing a significant step forward in understanding the arbuscular mycorrhizal fungus symbioses, also confirms that its optimal establishing and functioning might rely on many, as yet unidentified factors.

We screened cDNA libraries from periwinkle (Catharanthus roseus) cell cultures induced for indole alkaloid synthesis and selected clones for induced cytochrome P-450 (P-450) proteins by differential hybridization, size of the hybridizing mRNA, and presence of amino acid motifs conserved in many P-450 families. Four cDNAs satisfying these criteria were analyzed in detail. They were grouped in two classes (pCrosl, pCros2) that represented two closely related genes of a new P-450 family designated CYP72. Antiserum against a cDNA fusion protein overexpressed in Escherichia cofi recognized in C. roseus a protein band of 56 kD. Quantification of western blots showed that it represented 1.5 ± 0.5 and 6 ± 1 ag/mg of protein in the membranes from noninduced and induced cells, respectively, and analysis of the total P-450 content suggested that the cDNA-encoded protein was one of the dominant P-450 proteins. The pathway to indole alkaloids contains two known P-450 enzymes, geraniol-10-hydroxylase (GE1OH) and nerol-10-hydroxylase (NE1OH). The induction kinetics of the cloned P-450 protein and of GE10H activity were similar, but those of NE10H were different. Western blots with membranes from other plants suggested that P-450 CYP72 is specific for C. roseus and other plants with GE10H activity. A tentative assignment of CYP72 as GE1OH is discussed. The cDNA was recloned for expression in Saccharomyces cerevisiae, and the presence of the protein was demonstrated by western blots. Assays for GE1OH failed to detect enzyme activity, and the same negative result was obtained for NE10H and other P-450 enzymes that are present in C. roseus. P-4502 are the terminal oxidases of a large number of biotransformations. The enzymic reactions include metabo-1 This work was supported by the Fonds der Chemischen Industrie and Deutsche Forschungsgemeinschaft (SFB206).2Abbreviations: P-450, cytochrome(s) P-450; CA4H, cinnamicacid-4-hydroxylase; 14DM, lanosterol 14a-demethylase; FL3'H, flavonoid-3'-hydroxylase; FL3'5'H, flavonoid-3',5'-hydroxylase; GE1OH, geraniol-10-hydroxylase; LAH, lauric-acid-hydroxylase (in chain); NE1OH, nerol-10-hydroxylase; EROD, 7-ethoxyresorufin-0-deethylase; MX medium, growth medium; IM2 medium, alkaloid induction medium.lism of steroids, fatty acids, prostaglandins, leukotrienes, biogenic amines, pheromones, drugs, plant metabolites, and numerous other substances, including mutagens (19). The importance of these enzymes led in the last 10 years to a dramatic increase of molecular research in animal systems (20).The importance of P-450 enzymes in plants has been recognized (5), but the molecular analysis is lagging. The approach from purified protein to molecular biology has been difficult. In plants, the concentrations of these proteins are usually lower than in animals, the activities are unstable, the reconstitution of solubilized components to enzymically active complexes is difficult, and antisera against purified plant proteins or against P-450 enzymes from other sources most often recognize several...

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