RESUMEN

The effect of trace metal nutrition on the functioning of the patulin biosynthetic pathway in submerged cultures of Penicillium urticae (NRRL 2159A) was examined by both chromatographic and enzymological means. Comprehensive metal ion analysis showed generally low levels of contaminating metal ions in media components. Of eight metal ions examined, only manganese strongly influenced secondary metabolite production. In control cultures or cultures deficient in calcium, iron, cobalt, copper, zinc, or molybdenum, pathway metabolites appeared in the medium at about 25 h after inoculation. The first pathway-specific metabolite, 6-methylsalicylic acid, accumulated only transiently before being converted to patulin whose concentration steadily increased. In manganese-deficient cultures, however, 6-methylsalicylic acid continued to accumulate, with only minor amounts of patulin being produced. Additionally, a marker enzyme for the pathway showed only 0-20% of control activity. Clear dose responses (patulin versus manganese) were found in different media, with no effect on growth yield. Addition of manganese to depleted cultures at 18, 26, or 36 h resulted in increasing marker enzyme activity and patulin concentrations. It is concluded that manganese exerts a specific, positive effect on patulin biosynthesis and may in some way control the section of the patulin pathway occurring after 6-methylsalicylic acid.

Asunto(s)

RESUMEN

Two closely related species of mycobacteria, Mycobacterium vaccae and M. neoaurum, were grown under conditions of iron-deficiency (0.02-0.05 microgram Fe ml-1) and iron-sufficiency (2-4 micrograms Fe ml-1) in a simple glycerol/asparagine medium. The strain of M. vaccae used was a nonmycobactin producer whereas M. neoaurum synthesized between 6-8% of its cell biomass as the lipid-soluble siderophore when grown under iron-limitation. The role of mycobactin for iron-acquisition was examined using both pure and mixed cultures, with cell viability determined following growth at various iron concentrations. M. neoaurum, the mycobactin producer, outgrew M. vaccae when iron was readily available. When grown under conditions where iron was limiting, M. neoaurum showed a decline in viable cell number compared with its competitor, highlighting its increased requirement for the metal. Some recovery was observed following mycobactin biosynthesis, this being greatly enhanced by the addition of an iron supplement to the growing cells. Mycobactin biosynthesis allowed M. neoaurum to rapidly acquire any additional iron presented to the bacteria when growing under iron-limitation. However, M. vaccae did not synthesize the lipid-soluble siderophore with its iron-requirement satisfied by production of extracellular exochelin.

Asunto(s)

RESUMEN

The initiation of patulin biosynthesis in submerged batch cultures of Penicillium urticae NRRL 2159A was investigated at the enzyme level. In contrast to earlier studies, this study achieved a clear temporal separation of growing cells devoid of secondary metabolism-specific enzymes from nongrowing cells, which rapidly produce these enzymes. A spore inoculum, silicone-treated flasks, and two new media which supported a rapid, pellet-free, filamentous type of growth were used. In yeast extract-glucose-buffer medium, a marked drop in the specific growth rate (approximately equal to 0.26 h-1) coincided with the appearance of the first pathway-specific enzyme, 6-methylsalicylic acid synthetase, at about 19 h after inoculation. About 3 h later, when replicatory growth had ceased entirely, the sparsely branched mycelia (length, approximately equal to 550 microns) began the rapid synthesis of a later pathway enzyme, m-hydroxybenzyl alcohol dehydrogenase. A similar sequence of events occurred in a defined nitrate-glucose-buffer medium; 12 other strains or isolates of P. urticae, as well as some patulin-producing aspergilli, behaved in a similar manner. The age at which a culture produced m-hydroxybenzyl alcohol dehydrogenase was increased by increasing the nutrient nitrogen content of the medium or by decreasing the size of the spore inoculum. In each instance the appearance of enzyme was determined by the nutritional status of the culture and not by its age. A similar appearance of patulin pathway enzymes occurred when a growing culture was resuspended in a nitrogen-free 4% glucose solution with or without 0.1 M phosphate (pH 6.5). The appearance of both the synthetase and the dehydrogenase was arrested by the addition of cycloheximide (0.4 to 5 micrograms/ml) or actinomycin D (20 to 80 micrograms/ml). This requirement for de novo protein and ribonucleic acid syntheses was confirmed by the incorporation of labeled leucine into the dehydrogenase, and the possibility that latent or preformed proteins were being activated was eliminated.

Asunto(s)

RESUMEN

Reduction in the amounts of activity of the first enzyme, aspartokinase (EC 2.7.2.4) and two branch-point enzymes, dihydrodipicolinic acid synthase (EC 4.2.1.52) and homoserine dehydrogenase (EC 1.1.1.3), located in the pathway for the synthesis of aspartate-family amino acids, occurred when cell suspension cultures of Daucus carota L. var. Danvers were grown in media containing 2 mM threonine or 2 mM lysine, endproducts of the pathway. Activity of the lysine-sensitive form of aspartokinase was decreased when cells were grown in medium containing lysine and the activity of the threonine-sensitive form was decreased when cells were grown in medium containing threonine. Activity of the branch-point enzyme leading to threonine synthesis, homoserine dehydrogenase, was decreased up to 70% in specific activity (units/mg protein) and relative activity (units/g fresh weight) when cells were grown in media containing lysine or threonine. Threonine had no effect on the relative activity of dihydrodipicolinic acid synthase, but decreased its specific activity. Lysine decreased the relative activity of the synthase by up to 40%, but had little effect on its specific activity. The decreased activities of the enzymes were apparently not due to binding of the inhibitory amino acids to the enzymes since homogenization of cells in buffer with 2 mM lysine and threonine did not decrease the measurable enzyme activities. These and other results presented suggest that both forms of the aspartokinase activity and homoserine dehydrogenase activity can be altered by supplementing the growth medium with lysine or threonine.

Asunto(s)

Asunto(s)

RESUMEN

Anacardic (6-alkylsalicylic) acids and common lipids are efficiently synthesized by immature seeds of Ginkgo biloba. The seeds were incubated with 14C-labeled acetic, malonic and palmitoleic acids, glucose, and other potential precursors. Levels of 14C in common lipids and in anacardic acids, and the distribution of 14C in anacardic acids were determined. The results show that the salicylic moiety is synthesized by a polyketide pathway via malonic acid. The chain moiety for anacardic acid synthesis is in a different state of activation and/or site than chains that are used for synthesis of the common lipids. Labeled shikimic acid did not contribute 14C to anacardic acids, nor to other lipids, and palmitoleic acid was incorporated only into common lipids.

Asunto(s)

RESUMEN

Cerulenin, a specific inhibitor of beta-ketoacyl-(acyl carrier protein) synthetase [EC 2.3.1.41] and 3-hydroxy-3-methylglutaryl-CoA synthetase [EC 4.1.3.5], was studied to determine whether it inhibits 6-methylsalicylic acid synthesis, in which so-called "polyketide" formation, a condensation step similar to that in fatty acid synthesis, is involved. In fact, 100 mug/ml (4.5 X 10(-4) M) of cerulenin inhibited 60% of 6-methylsalicylic acid synthetase activity and 68% of fatty acid synthetase activity of Penicillium urticae.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

The objectives of this research were to determine the kinetics of salicylate elimination in anephric patients and particularly to establish if these patients form the major metabolite of salicylic acid, salicyluric acid, at a normal rate. This investigation was initiated because of conflicting reports concerning the contribution of the kidneys to the formation of salicyluric acid in man. Six patients, 20-44 yr old, three of whom were anatomically anephric while the other three were physiologically anephric, received an intravenous injection of 500 mg salicylic acid (as sodium salicylate)/1.73 m(2) body surface area on an interdialysis day. Serial blood samples were obtained for 12 or 16 h after injection and the plasma was assayed for salicylic acid, salicyluric acid, total protein, albumin, and creatinine. Detailed pharmacokinetic analysis based on an open, two-compartment linear model revealed no significant differences in apparent volume of distribution and apparent first-order distribution and elimination rate constants between the anephric patients and normal adult subjects. An estimate of salicyluric acid formation rate by the anephric patients, based on the initial rate of increase of salicylurate concentrations in plasma, indicates that the metabolite is formed at a normal rate. These results suggest that the kidneys do not contribute significantly to the formation of salicyluric acid from salicylic acid in man.

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

Mycobacterium smegmatis was grown on trace-metal-free medium in static culture. Throughout the growth phase, the concentration of mycobactin increased continuously, reaching a maximum of about 30 to 40 mug of mycobactin/mg of cell dry weight after 6 days; the concentration of salicylic acid remained approximately constant at 1 to 2 mug of salicylic acid/mug of cell dry weight. Fe(2+) (or Fe(3+)), Zn(2+), Mn(2+), and Mg(2+) were all essential to a maximum formation of mycobactin. Optimum concentrations required were: Fe(2+), about 1.8 mum; Mn(2+) and Zn(2+), about 0.5 mum; and Mg(2+), at least 0.17 mm. Higher levels of Fe(2+) (9 to 90 mum) and Zn(2+) (2 to 7 mum) repressed mycobactin to about half the maximum value. No other cation or anion apparently is required for mycobactin biosynthesis. Salicylic acid concentration increased about fourfold when iron was omitted from the medium, but this is not as great as the increase reported previously for this strain of M. smegmatis. Mycobactin formation in another strain of M. smegmatis, NCIB 8548, showed similar dependencies on Fe(2+), Zn(2+), and Mn(2+). Maximum accumulation of mycobactin with this strain was 85 mug of mycobactin/mg of dry cell weight, under iron-deficient (1.8 mum Fe(2+)) conditions.

Asunto(s)

Asunto(s)