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Biomedical subjects

J A Gil

Publications and source records attributed to J A Gil.

46 records · Page 3Linked to original sources
PubMed1989

Sporulation of several species of Streptomyces in submerged cultures after nutritional downshift.

Streptomyces griseus ATCC 10137, S. griseus IMRU 3570, S. griseus JI 2212, S. acrimycini JI 2236 and S. albus G sporulated abundantly in several liquid media after nutritional downshift. Spores formed in submerged cultures were viable and as thermoresistant as aerial spores. Scanning electron microscopy showed that submerged spores are morphologically similar to aerial spores. The sporulation of the Streptomyces strains tested in complex medium appeared to be triggered by phosphate nutritional downshift, induced by addition of Ca2+ to the medium. Spore-shaped bodies were formed by S. lividans JI 1326 and S. coelicolor JI 2280 when grown in complex medium supplemented with Ca2+ and proline. The thermoresistance of these spore-shaped bodies differed from that of aerial spores.

Calcium Chloride↗
PubMed1987

Identification of a promoter sequence in the plasmid pUL340 of Brevibacterium lactofermentum and construction of new cloning vectors for corynebacteria containing two selectable markers.

A strong promoter P1 has been found in plasmid pUL340, a cloning vector used to transform corynebacteria. This promoter is also expressed efficiently in Escherichia coli. A gene (cat) for chloramphenicol acetyltransferase from Streptomyces acrimycini and a gene (hyg) for hygromycin phosphotransferase from Streptomyces hygroscopicus were subcloned in different positions of the Brevibacterium lactofermentum plasmid pUL340. Both resistance genes are expressed in B. lactofermentum from their own promoters or from the endogenous promoter in pUL340. These genes provide useful screening markers for selecting transformants of B. lactofermentum together with the kanamycin-resistance gene from the transposon Tn5.

Brevibacterium↗
PubMed1985

Cloning of a chloramphenicol acetyltransferase gene of Streptomyces acrimycini and its expression in Streptomyces and Escherichia coli.

A gene (cat) for chloramphenicol (Cm) acetyltransferase (CAT) was cloned from Streptomyces acrimycini into S. lividans 66 on the plasmid vector pIJ61. The cat gene was localized on a 1.7-kb BclI fragment, which probably also carries the cat promoter. This DNA fragment conferred Cm resistance, through CAT activity, on S. lividans, S. coelicolor and S. parvulus, but not on Escherichia coli when inserted in the BamHI site of the tetracycline-resistance(TcR) gene of pBR322. However, when inserted in a particular orientation in this site, spontaneous deletions of 0.7 kb led to CAT activity and Cm resistance. DNA homologous to the 1.7-kb BclI cat fragment was found in most, but not all, of a series of other streptomycetes that have CAT activity. The cat provides a potentially useful screening marker for Streptomyces cloning vectors.

Acetyltransferases↗
PubMed1985

Characterization and regulation of p-aminobenzoic acid synthase from Streptomyces griseus.

p-Aminobenzoic acid synthase (PABA synthase) of Streptomyces griseus catalyses the conversion of chorismic acid to p-aminobenzoic acid (PABA), a precursor of the aromatic p-aminoacetophenone moiety of candicidin, a polyene macrolide antibiotic. This enzyme uses glutamine or ammonia as amino donors for PABA formation. Enzyme extracts converted [14C]chorismic acid to labelled PABA. PABA synthase was present in S. griseus IMRU 3570 only during the antibiotic producing phase. No detectable levels of the enzyme were found in cell-free extracts of nonproducing mutants of S. griseus obtained after UV mutagenesis. PABA synthase activity was found also in Streptomyces coelicolor var. aminophilus, producer of the polyene macrolide antibiotic fungimycin, but it was not present in extracts of several other streptomycetes that do not produce aromatic polyene macrolide antibiotics. PABA synthase (amidotransferase) activity was partially purified by DEAE-Bio-gel and Sephacryl S-200 filtrations. The estimated molecular weight was 50000. PABA synthase was repressed by aromatic amino acids and PABA but not by anthranilic acid. Inorganic phosphate strongly repressed but did not inhibit PABA synthase activity.

Amino Acids↗
PubMed1985

High-frequency transformation of Brevibacterium lactofermentum protoplasts by plasmid DNA.

An efficient polyethylene glycol-assisted method for transformation of Brevibacterium lactofermentum protoplasts that uses plasmid vectors has been developed. Two small plasmids, pUL330 (5.2 kilobases) and pUL340 (5.8 kilobases), both containing the kanamycin resistance gene from transposon Tn5 and the replication origin of the natural plasmid pBL1 of B. lactofermentum, were selected as vectors. Supercoiled forms of the plasmids yielded a 100-fold higher transformation frequency than did linear forms. The optimal transformation frequency was achieved with 10 ng of DNA in 1 ml of transformation buffer. Higher concentrations of plasmid DNA resulted in a decrease in transformation frequency per microgram of DNA. Optimal transformation was obtained with 25 to 35% polyethylene glycol 6000. Under optimal conditions, 10(6) transformants per microgram of DNA were obtained.

Brevibacterium↗
PubMed1983

Cloning and expression of a p-aminobenzoic acid synthetase gene of the candicidin-producing Streptomyces griseus.

4.5-kb BamHI fragments of DNA coding for p-aminobenzoic acid (PABA) synthetase from the candicidin-producing Streptomyces griseus IMRU 3570 and from a sulphonamide resistant mutant of it were cloned on the plasmid vector pIJ41 into Streptomyces lividans 66. The cloned DNA restored prototrophy to a pab auxotroph of S. lividans; when the S. griseus donor was a sulphonamide resistant, PABA-overproducing mutant, the S. lividans clone was sulphonamide resistant as well as Pab+. Sub-cloning the 4.5-kb fragment of S. griseus DNA into Escherichia coli pabA- or pabB- mutants by insertion at the BamHI site of pBR322 did not yield prototrophic clones directly. However, when the cloned fragment had the proper orientation relative to the tet promoter, but not the opposite one, it was possible to select Pab+ colonies, which arose by deletion in vivo of approx. 1 kb of the S. griseus inserted DNA. These results, and those of studies in which Tn5 abolished the Pab+ phenotype by insertion in vivo in the tet promoter or downstream of it, indicated that the S. griseus pab promoter was not expressed in E. coli but that the pab gene could be expressed by transcriptional readthrough from the vector. Experiments in which the cloned DNA was transferred back from E. coli to S. lividans suggested, but did not prove, that the Streptomyces pab promoter had been deleted by loss of the approx. 1-kb segment. These experiments showed expression of both the tet (of pBR322) and kan (of Tn5) promoters in S. lividans.

Antifungal Agents↗
PubMed1980

Regulation by aromatic amino acids of the biosynthesis of candicidin by Streptomyces griseus.

The biosynthesis by Streptomyces griseus of candicidin, an aromatic polyene macrolide antibiotic, was inhibited by L-tryptophan, L-phenylalanine and, to a lesser degree, by L-tyrosine. A mixture of the three aromatic amino acids inhibited candicidin biosynthesis to a greater extent than did each amino acid separately. L-Tryptophan strongly inhibited the incorporation of the labelled precursors propionate or 4-aminobenzoic acid into candicidin. Incorporation of propionate into candicidin was 50% inhibited by 2.5 mM-tryptophan. Inhibition by tryptophan did not require protein synthesis as the same effect was observed in cells in which protein synthesis was prevented by chloramphenicol. The inhibitory effect of L-tryptophan was partially reversed by exogenous 4-aminobenzoic acid suggesting that this effect is exerted at the level of 4-aminobenzoic acid synthase.

Antifungal Agents↗