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L C Vining

Publications and source records attributed to L C Vining.

At least 55 records · Page 3Linked to original sources
PubMed1989

Measurement of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase activity in Streptomyces clavuligerus by high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde.

A procedure for rapid, sensitive measurement of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) in complex cell extracts has been developed by adapting a widely used method for high-performance liquid chromatography analysis of amino acids. Samples were oxidized with performic acid and derivatized with o-phthaldialdehyde-mercaptoethanol to give the fluorescent isoindole derivative of the peptide sulfonate. The procedure was used to assay Streptomyces clavuligerus cell extracts and partially purified fractions for ACV synthetase activity and to determine some characteristics of the enzyme reaction. The presence of a second enzyme with cysteine as a substrate was also indicated.

Chromatography, High Pressure Liquid↗
PubMed1989

Culture conditions promoting dispersed growth and biphasic production of actinorhodin in shaken cultures of Streptomyces coelicolor A3(2).

Media and culture conditions were developed for experiments on the physiology of secondary metabolism in Streptomyces coelicolor A3(2). Well dispersed mycelial growth was obtained in a buffered starch-glutamate-salts medium; a high (5%) starch concentration and addition of glass beads aided dispersal. Under the conditions developed, production of actinorhodin was suppressed during trophophase growth and began abruptly near the growth maximum.

Anthraquinones↗
PubMed1989

Purification, properties and immunological detection of a bromoperoxidase-catalase from Streptomyces venezuelae and from a chloramphenicol-nonproducing mutant.

A new bromoperoxidase-catalase was purified from the chloramphenicol-producing actinomycete Streptomyces venezuelae ISP 5230. The homogeneous enzyme showed brominating activity, catalase activity and a very low peroxidase activity. The spectral properties and pH dependence of the catalase activity showed similarities to conventional catalases. In contrast to other haem-bromoperoxidases, the bromoperoxidase-catalase was stable when treated with an ethanol/chloroform mixture. Gel filtration gave an estimated Mr of 127,000-136,000. SDS-PAGE showed a single band corresponding in mobility to a species with an Mr of 61,000. The pI was estimated to be 4.5. The bromoperoxidase-catalase was not present in active form in a mutant of S. venezuelae ISP 5230, blocked in the chlorination step of chloramphenicol biosynthesis. However, an inactive species of the enzyme was detected in crude extracts of the mutant by using antibodies. From these results it is concluded that this bromoperoxidase participates in the chlorination step during chloramphenicol biosynthesis.

Catalase↗
PubMed1989

Lysine catabolism in Streptomyces spp. is primarily through cadaverine: beta-lactam producers also make alpha-aminoadipate.

Genetic and biochemical evidence was obtained for lysine catabolism via cadaverine and delta-aminovalerate in both the beta-lactam producer Streptomyces clavuligerus and the nonproducer Streptomyces lividans. This pathway is used when lysine is supplied as the sole source of nitrogen for the organism. A second pathway for lysine catabolism is present in S. clavuligerus but not in S. lividans. It leads to alpha-aminoadipate, a precursor for beta-lactam biosynthesis. Since it does not allow S. clavuligerus to grow on lysine as the sole nitrogen source, this pathway may be used exclusively to provide a precursor for beta-lactam biosynthesis. beta-Lactam producers were unable to grow well on alpha-aminoadipate as the only nitrogen source, whereas three of seven species not known to produce beta-lactam grew well under the same conditions. Lysine epsilon-aminotransferase, the initial enzyme in the alpha-aminoadipate pathway for lysine catabolism, was detected in cell extracts only from the beta-lactam producers. These results suggest that synthesis of alpha-aminoadipate is exclusively a secondary metabolic trait, present or expressed only in beta-lactam producers, while genes governing the catabolism of alpha-aminoadipate are present or fully expressed only in beta-lactam nonproducers.

2-Aminoadipic Acid↗
PubMed1988

Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus.

The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.

Acremonium↗
PubMed1988

Role of the carbon source in regulating chloramphenicol production by Streptomyces venezuelae: studies in batch and continuous cultures.

Both carbon- and nitrogen-limited media that supported a biphasic pattern of growth and chloramphenicol biosynthesis were devised for batch cultures of Streptomyces venezuelae. Where onset of the idiophase was associated with nitrogen depletion, a sharp peak of arylamine synthetase activity coincided with the onset of antibiotic production. The specific activity of the enzyme was highest when the carbon source in the medium was also near depletion at the trophophase-idiophase boundary. In media providing a substantial excess of carbon source through the idiophase, the peak specific activity was reduced by 75%, although the timing of enzyme synthesis was unaltered. Moreover, chemostat cultures in which the growth rate was limited by the glucose concentration in the input medium failed to show a decrease in specific production of chloramphenicol as the steady-state intracellular glucose concentration was increased. The results suggest that a form of "carbon catabolite repression" regulates synthesis of chloramphenicol biosynthetic enzymes during a trophophase-idiophase transition induced by nitrogen starvation. However, this regulatory mechanism does not establish the timing of antibiotic biosynthesis and does not function during nitrogen-sufficient growth in the presence of excess glucose.

Carbon↗
PubMed1987

Transductional analysis of chloramphenicol biosynthesis genes in Streptomyces venezuelae.

Auxotrophs isolated from two chloramphenicol-nonproducing mutants of Streptomyces venezuelae included three requiring pyridoxal (Pxl-), VS248 (cml-11 pdx-2), VS253 (cml-11 pdx-3), and VS258 (cml-12 pdx-4), and one requiring thiosulfate, VS263 (cml-12 cys-28). Results of SV1-mediated transductions were consistent with the relative marker order cys-28-cml-12-cml-11-pdx-2,3,4,5, all of which were cotransducible and must therefore span less than 45 kilobases of DNA, the approximate length of DNA packaged by SV1. cys-28 was also cotransducible with arg-4 and arg-6, but arg and pdx were not cotransducible. Results of crosses with donors carrying any one of 11 cml mutations were consistent with the location of all cml mutations between cys-28 and pdx markers. Also, a new Pxl- auxotroph (pdx-6) and two new Cml- mutants were recovered after localized hydroxylamine mutagenesis of a cys-28 cml+ strain derived from VS263 by transduction.

Chloramphenicol↗
PubMed1987

Characterization of new viridomycins and requirements for production in cultures of Streptomyces griseus.

Cultures of Streptomyces griseus grown under phosphate-limiting conditions produced a complex of green products. Three of these were separated from the mixture and characterized. One was identified as viridomycin A, the ferrous chelate of 4-hydroxy-3-nitrosobenzaldehyde; the second (actinoviridin A) was the corresponding carboxylic acid chelate and the third (viridomycin E) was a hybrid chelate containing both the aldehyde and acid ligands. Only two out of nine strains of S. griseus examined produced viridomycins and the ligands were biosynthesized only in media from which phosphate had been exhausted. Optimization of the production medium showed that fructose and alanine were the most favorable carbon and nitrogen sources and that relatively high concentrations of ferrous ions were necessary. The results suggest that viridomycins are not produced by S. griseus as iron scavengers in response to iron deficiency but as secondary metabolites that are stabilized adventitiously in the broth by metal ion chelation.

Anti-Bacterial Agents↗
PubMed1986

Conjugational fertility and location of chloramphenicol biosynthesis genes on the chromosomal linkage map of Streptomyces venezuelae.

In Streptomyces venezuelae fertility, defined as chromosomal gene recombination, was enhanced over 1000-fold when one parent in a biparental conjugational cross lacked the physically-undetected plasmid SVP1, as compared with crosses in which both parents carried SVP1. The existence of SVP1 and at least two other fertility plasmids, SVP2 and SVP3, was detected in S. venezuelae by 'lethal zygosis' elicited by a plasmid-plus mycelium in contact with a plasmid-minus mycelium. Conjugational crosses were used to construct a linkage map of S. venezuelae which was highly consistent with the map of analogous loci in S. coelicolor A3(2). A cluster of genes governing chloramphenicol biosynthesis was located near arg, cys and pdxB genes at a position roughly equivalent to the 1-2 o'clock region of the S. coelicolor A3(2) map.

Chloramphenicol↗
PubMed1986

Purification of isopenicillin N synthetase from Streptomyces clavuligerus.

Isopenicillin N synthetase was purified from Streptomyces clavuligerus by sequential salt precipitation, ion-exchange and gel-filtration chromatography using both conventional open column and high-performance liquid chromatographic techniques. Material from the final purification step had a specific activity of 204.1 X 10(-3) units/mg of protein which represented a 130-fold purification over the cell-free extract. The purified isopenicillin N synthetase was determined to have a molecular weight of 33,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and to have a Km of 0.32 mM with respect to its substrate delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine. The enzyme showed a sensitivity to thiol-specific inhibitors with N-ethylmaleimide giving the strongest inhibitory effect.

Chromatography, Gel↗
PubMed1986

Use of carbon-13 in biosynthetic studies: origin of the malonyl coenzyme A incorporated into tetracycline by Streptomyces aureofaciens.

The proton noise decoupled 13C nuclear magnetic resonance spectrum of tetracycline hydrochloride prepared from Streptomyces aureofaciens cultures supplemented with [1-13C]acetate and [2-13C]acetate showed enrichment of nine alternating ring carbons. In addition, a small enrichment of the carboxamide carbon by [1-13C]acetate was observed. The labelling patterns clearly demonstrated the polyketide origin of the tetracyclic nucleus. The 13C nuclear magnetic resonance spectrum of tetracycline hydrochloride derived from [1,2-13C]acetate showed all 18 ring carbons as doublets with coupling constants appropriate for the incorporation of nine intact two-carbon precursors, confirming that head-to-tail condensation of C2 units had occurred. Absence of bond scission within the C2 units and a low level of uncoupled 13C in the carboxamide substituent indicated that when the organism is supplemented with acetate, malonyl coenzyme A used for tetracycline biosynthesis is formed by direct carboxylation of acetyl coenzyme A.

Acetates↗
PubMed1985

Isolation and characterization of Streptomyces venezuelae mutants blocked in chloramphenicol biosynthesis.

Twelve Streptomyces venezuelae mutants blocked in chloramphenicol biosynthesis were isolated. Two of these (Cm1-1 and Cm1-12) were apparently blocked in the conversion of chorismic acid to p-aminophenylalanine and three (Cm1-4, Cm1-5 and Cm1-8) accumulated p-aminophenylalanine and may have been blocked in the hydroxylation reaction that converted this intermediate to p-aminophenylserine. One mutant (Cm1-2) accumulated D-threo-1-p-nitrophenyl-2-propionamido-1,3-propanediol and D-threo-1-p-nitrophenyl-2-isobutyramido-1,3-propanediol, indicating that chlorination of the alpha-N-acyl group of chloramphenicol was blocked. The remaining six strains did not excrete any detectable chloramphenicol pathway intermediates.

Chloramphenicol↗
PubMed1985

Effect of ammonium on chloramphenicol production by Streptomyces venezuelae in batch and continuous cultures.

Cultures of Streptomyces venezuelae presented with a mixture of ammonium and an amino acid as nitrogen sources used both compounds together. Absence of ammonium repression of alternative nitrogen assimilation pathways was also observed when ammonium was added to cultures already growing on proline. The presence of ammonium in the medium ab initio depressed the yield of chloramphenicol. However, its addition to a culture growing on proline caused only a temporary inhibition of antibiotic synthesis, even when sufficient ammonium was added to create an excess. Continuous cultures supplied with ammonium as the growth-limiting nutrient showed no significant change in specific antibiotic production at different specific growth rates. The overall results indicate that in S. venezuelae neither nitrogen utilization pathways nor chloramphenicol biosynthesis is controlled by nitrogen repression.

Bacteriological Techniques↗
PubMed1985

Pathway of ammonium assimilation in Streptomyces venezuelae examined by amino acid analyses and 15N nuclear magnetic resonance spectroscopy.

To obtain information on the route(s) of ammonium assimilation in Streptomyces venezuelae, cell suspensions transferred to fresh medium lacking nitrogen were pulsed with [15N2]ammonium sulphate. Cells and extracellular fluids were examined by nuclear magnetic resonance and amino acid analysis to assess changes in amino acid pools and the disposition of [15N]ammonium. Following addition of [15N]ammonium, glutamate--glutamine pools of low cell density replacement cultures expanded rapidly and became progressively labelled with 15N, whereas the alanine pool size increased much more slowly and became labelled with 15N to a much lesser extent. These results are consistent with the assimilation of ammonium via glutamate dehydrogenase or glutamine synthetase--glutamate synthase rather than alanine dehydrogenase. Under anaerobic conditions, S. venezuelae assimilates ammonium into alanine rather than glutamate--glutamine. Alanine dehydrogenase may thus function as a vehicle to regenerate NAD+ to maintain substrate-level phosphorylation during periods of anaerobiosis.

Alanine↗
PubMed1984

Suppression of nitrate utilization by ammonium and its relationship to chloramphenicol production in Streptomyces venezuelae.

Cultures of Streptomyces venezuelae grown in a medium containing glucose with mixtures of ammonium and nitrate as the nitrogen source produced chloramphenicol in a distinct idiophase that followed biomass accumulation. Analysis of fermentation broths showed that ammonium and nitrate were taken up consecutively by the organism. Measurements of nitrate reductase in the mycelium established that the enzyme was constitutive and that its specific activity did not increase during the period when ammonium was exhausted from the medium and nitrate was assimilated. The enzyme was neither repressed nor inhibited by ammonium. Production of chloramphenicol was also delayed until ammonium had been consumed and remained slow until subsequent depletion of nitrate. Arylamine synthetase, the initial enzyme in the pathway of antibiotic biosynthesis, showed no marked change in specific activity during utilization of the two nitrogen sources. The result suggests that the mechanism causing preferential utilization of ammonium does not simultaneously control the onset of chloramphenicol biosynthesis.

Chloramphenicol↗
PubMed1984

Excretion of alpha-keto acids by strains of Streptomyces venezuelae.

Cultures of Streptomyces venezuelae released acidic metabolites during nitrogen-limited growth on glucose. The main products were pyruvic acid and alpha-ketoglutaric acid. Variation in the extent of acid production was observed; spores of the parental strain 13s gave approximately 10% of low-producing colonies when plated on acid-base indicator medium. Examination of one low producer, strain PC 51-5, showed that differences in acid production became apparent only in low-glucose media containing manganese. In both strains PC 51-5 and 13s, uptake of alpha-keto-[5-14C]glutaric acid occurred by diffusion and no marked differences in permeability to alpha-ketoglutarate were detected. However, differences were observed in the activity of alpha-ketoglutarate dehydrogenase. In cultures of strain PC 51-5, the specific activity of the enzyme increased throughout growth, whereas in the parental strain activity decreased and could not be detected in older mycelium. Loss of enzyme activity was accompanied by excretion of alpha-ketoglutaric acid and failure to assimilate the product after glucose exhaustion. The results suggest that accumulation of pyruvic and alpha-ketoglutaric acids in S. venezuelae cultures grown in glucose-containing media may be due to regulatory suppression of the dehydrogenases by this carbon source.

Cell Membrane Permeability↗