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P Liras

Publications and source records attributed to P Liras.

At least 55 records · Page 3Linked to original sources
PubMed1991

A gene encoding lysine 6-aminotransferase, which forms the beta-lactam precursor alpha-aminoadipic acid, is located in the cluster of cephamycin biosynthetic genes in Nocardia lactamdurans.

A gene (lat) encoding lysine 6-aminotransferase was found upstream of the pcbAB (encoding alpha-aminoadipylcysteinyl-valine synthetase) and pcbC (encoding isopenicillin N synthase) genes in the cluster of early cephamycin biosynthetic genes in Nocardia lactamdurans. The lat gene was separated by a small intergenic region of 64 bp from the 5' end of the pcbAB gene. The lat gene contained an open reading frame of 1,353 nucleotides (71.4% G + C) encoding a protein of 450 amino acids with a deduced molecular mass of 48,811 Da. Expression of DNA fragments carrying the lat gene in Streptomyces lividans led to a high lysine 6-aminotransferase activity which was absent from untransformed S. lividans. The enzyme was partially purified from S. lividans(pULBS8) and showed a molecular mass of 52,800 Da as calculated by Sephadex gel filtration and polyacrylamide gel electrophoresis. DNA sequences which hybridized strongly with the lat gene of N. lactamdurans were found in four cephamycin-producing Streptomyces species but not in four other actinomycetes which are not known to produce beta-lactams, suggesting that the gene is specific for beta-lactam biosynthesis and is not involved in general lysine catabolism. The protein encoded by the lat gene showed similarity to ornithine-5-aminotransferases and N-acetylornithine-5-aminotransferases and contained a pyridoxal phosphate-binding consensus amino acid sequence around Lys-300 of the protein. The evolutionary implications of the lat gene as a true beta-lactam biosynthetic gene are discussed.

2-Aminoadipic Acid↗
PubMed1991

Characterization, expression in Streptomyces lividans, and processing of the amylase of Streptomyces griseus IMRU 3570: two different amylases are derived from the same gene by an intracellular processing mechanism.

Extracellular amylase in Streptomyces lividans was undetectable in starch-supplemented medium. However, S. lividans produced fivefold-higher levels of amylase than Streptomyces griseus IMRU 3570 when transformed with the S. griseus amy gene. Two major proteins of 57 and 50 kDa with amylase activity accumulated in the culture broths of the donor S. griseus and S. lividans transformed with the amy gene. Both proteins were also present in protoplast lysates in the same relative proportion; they gave a positive reaction with antibodies against the 57-kDa amylase. They did not differ in substrate specificity or enzyme kinetics. The two amylases were purified to homogeneity by a two-step procedure. Both proteins showed the same amino-terminal sequence of amino acids, suggesting that both proteins are derived from the same gene. The deduced signal peptide has 28 amino acids with two positively charged arginines near the amino-terminal end. When an internal NcoI fragment was removed from the amy gene, the resulting S. lividans transformants did not synthesize any of the two amylase proteins and showed no reaction in immunoblotting. Formation of the 50-kDa protein was observed when pure 57-kDa amylase was treated with supernatants of protoplast lysates but not when it was treated with membrane preparations, indicating that the native 57-kDa amylase could be processed intracellularly.

Amino Acid Sequence↗
PubMed1990

Phosphate control of pabS gene transcription during candicidin biosynthesis.

The pabS gene of Streptomyces griseus IMRU3570 encodes the enzyme p-aminobenzoic acid synthase, which synthesizes p-aminobenzoic acid (PABA), a precursor of the antibiotic candicidin (Cd). The pabS transcript reached a peak at 12 h of incubation in batch cultures, preceding the formation of PABA synthase and the antibiotic itself. A decay of the pabS transcript was observed with an apparent half-life of 35 min. Inorganic phosphate (Pi; 7.5 mM) reduced the synthesis of the pabS transcript by 90-95%, and consequently the formation of PABA synthase and Cd. Thirty min after addition of 7.5 mM Pi, the cells synthesized only about 15% as much pabS transcript compared to control cultures. However, Pi stimulated two- to threefold total RNA synthesis. The 1.7-kb pabS transcript shown by Northern hybridization was greatly reduced in amount in cells grown in 7.5 mM phosphate. Pi-deregulated mutants, described previously, were impaired in the transcriptional control exerted by Pi. It is concluded that Pi control of PABA synthase and Cd biosynthesis is exerted by repression of formation of the pabS mRNA.

Blotting, Northern↗
PubMed1990

A bifunctional Streptomyces-E. coli promoter-probe vector.

A bifunctional Streptomyces-E. coli promoter probe vector, pULJA30, has been developed to isolate and characterize nucleotide sequences involved in transcription initiation and regulation. The vector is derived from plasmid pIJ486, carries the pIJ101 replicon and utilizes the promoterless aminoglycoside phosphotransferase (neo) as indicator gene. Important features of the new vector include: wide Streptomyces host range and as high a plasmid copy number as the parental pIJ486, an upstream transcriptional terminator (toop) and a polylinker sequence with unique sites for BamHI and BglII for flexible cloning, fragment re-isolation and direct sequencing of promoter-active inserts. pULJA30 also has an E. coli replicon (from pBR322) and the possibility of selection in Streptomyces and E. coli by using the tsr, neo and bla genes, which makes it very convenient to test the comparative functionality of Streptomyces promoters in E. coli.

Base Sequence↗
PubMed1990

Phosphate control sequences involved in transcriptional regulation of antibiotic biosynthesis.

The expression of genes encoding enzymes involved in antibiotic and other secondary metabolite biosynthesis is down-regulated by easily assimilable phosphate, carbon and nitrogen sources. Phosphate control of antibiotic production appears to act at the transcriptional level by a mechanism similar to that involved in control of phosphatases and other phosphate-regulated enzymes. A phosphate control (PC) sequence, strikingly similar to the phosphate control (pho) boxes of many bacterial genes, has been isolated from the phosphate regulated promoter that controls biosynthesis of the antibiotic candicidin, and characterized. From computer analysis of sequence data, PC sequences appear to be associated with promoter regions of several phosphate-controlled antibiotic biosynthetic genes.

Anti-Bacterial Agents↗
PubMed1989

Cloning and characterization of a phosphate-regulated promoter involved in phosphate control of candicidin biosynthesis.

Phosphate strongly repressed the formation of p-aminobenzoic acid (PABA) synthase, an enzyme involved in candicidin biosynthesis. Expression in Streptomyces lividans of the pabS gene (encoding PABA synthase) of Streptomyces griseus is repressed by phosphate at concentrations above 0.1 mM. However, expression of the pabS gene in Escherichia coli is not regulated by phosphate. Phosphate control of the expression of the pabS gene was observed in all plasmids containing the original 4.5-kb BamHI fragment, whereas no phosphate regulation was found when an upstream 1-kb fragment that carries the pabS promoter was deleted. Using the promoter-probe plasmid pIJ424, a '114-bp' promoter was cloned. Expression of the promoterless kanamycin phosphotransferase gene when fused to the '114-bp' promoter was strongly reduced by phosphate (90% at 5 mM concentration). The '114-bp' promoter has been sequenced and the first transcribed nucleotide identified by S1 mapping. The '114-bp' fragment is A + T-rich (54%), as compared to the Streptomyces genome (70-73% GC). The presence of a phosphate control sequence (pcs) in the upstream region of the pabS gene is proposed.

Antifungal Agents↗
PubMed1989

Characterization of sugar uptake in wild-type Streptomyces clavuligerus, which is impaired in glucose uptake, and in a glucose-utilizing mutant.

Wild-type Streptomyces clavuligerus NRRL 3585 is unable to utilize glucose. A glucose-utilizing (gut-1) mutant of S. clavuligerus NRRL 3585 has been obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The gut-1 mutant is able to grow on glucose or galactose, while the wild type is unable to catabolize these hexoses. Similar binding affinities of glucose by cells of the wild type and the gut-1 mutant were found, but the wild type was unable to complete glucose transport. A soluble intracellular ATP-dependent (but not phosphoenolpyruvate-dependent) glucokinase activity was found both in the wild type and the gut-1 mutant. The gut-1 mutant has acquired a functional transport system that allows transport of glucose, 2-deoxyglucose, and galactose, as shown by hexose competition experiments. The gut-1 transport system concentrates glucose inside the cell at least 10- to 20-fold and is strongly inhibited by respiratory inhibitors, which prevent the establishment of a proton motive force, and by proton-conducting ionophores, suggesting that it is energized by a proton motive force. The new transport system is not completely sugar specific (transporting galactose and glucose through the same system), as opposed to the hexose-specific system reported in wild-type Streptomyces griseus.

Biological Transport, Active↗
PubMed1988

Purification and characterization of the isopenicillin N synthase of Streptomyces lactamdurans.

The isopenicillin N synthase (cyclase) of Streptomyces lactamdurans (syn. Nocardia lactamdurans) has been purified to near homogeneity as judged by SDS-PAGE and isoelectric focusing. This enzyme catalyses the oxidative cyclization of the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. The enzyme required DTT, Fe2+ and oxygen and it was greatly stimulated by ascorbic acid. It was strongly inhibited by Co2+, Zn2+ and Mn2+. Optimal pH and temperature were 7.0 and 25 degrees C (with the assay conditions used), respectively. The apparent Km of isopenicillin N synthase for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine was 0.18 mM. The enzyme is a monomer with an Mr of 26,500 +/- 1000 and a pI of 6.55.

Chromatography, Gel↗
PubMed1987

Purification and characterization of a 2-oxoglutarate-linked ATP-independent deacetoxycephalosporin C synthase of Streptomyces lactamdurans.

The deacetoxycephalosporin C (DAOC) synthase (expandase) of Streptomyces lactamdurans was highly purified, as shown by SDS-PAGE and isoelectric focusing. The enzyme catalysed the oxidative ring expansion that converts penicillin N into DAOC. The enzyme was very unstable but could be partially stabilized in 25 mM-Tris/HCl, pH 9.0, in the presence of DTT (0.1 mM). The enzyme required 2-oxoglutarate, oxygen and Fe2+, but did not need ATP, ascorbic acid, Mg2+ or K+. The optimum temperature was between 25 and 30 degrees C. The DAOC synthase showed a high specificity for the penicillin substrate. Only penicillin N but not isopenicillin N, penicillin G or 6-aminopenicillanic acid served as substrates. 2-Oxoglutarate analogues were not used as substrates although 2-oxobutyrate and 3-oxoadipate inhibited the enzyme by 100% and 56% respectively. The enzyme was strongly inhibited by Cu2+, Co2+ and Zn2+. The apparent Km values for penicillin N, 2-oxoglutarate and Fe2+ were 52 microM, 3 microM and 71 microM respectively. The enzyme was a monomer with a molecular mass of 27,000 Da +/- 1,000.

Chromatography, Gel↗
PubMed1987

Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus.

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.

Anti-Bacterial Agents↗
PubMed1986

Glucose regulation of cephamycin biosynthesis in Streptomyces lactamdurans is exerted on the formation of alpha-aminoadipyl-cysteinyl-valine and deacetoxycephalosporin C synthase.

Glucose exerted a concentration-dependent negative regulation on the biosynthesis of cephamycin C by Streptomyces lactamdurans. Formation of the cephamycin precursor delta(alpha-aminoadipyl)-cysteinyl-valine was greatly decreased by excess glucose. The ring-expanding enzyme deacetoxycephalosporin C synthase was strongly repressed by glucose in vivo. Isopenicillin N synthase (cyclase) and isopenicillin N epimerase were not repressed by glucose. However, the activity of isopenicillin N synthase was inhibited in vitro by glucose 6-phosphate, and the activity of deacetoxycephalosporin C synthase was inhibited by inorganic phosphate, glucose 6-phosphate, fructose 2,6-diphosphate and fructose 1,6-diphosphate. The intracellular cAMP content decreased as growth proceeded and remained lower in glucose-supplemented cells than in control cultures. cAMP did not seem to be involved in glucose control of cephamycin biosynthesis.

Amino Acid Isomerases↗
PubMed1986

Utilization of ornithine and arginine as specific precursors of clavulanic acid.

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.

Arginine↗
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↗
PubMed1979

Isolation of mutants deregulated in phosphate control of candicidin biosynthesis.

Mutants have been isolated in which phosphate does not inhibit the biosynthesis of candicidin. At high phosphate concentrations, candicidin production by phosphate-deregulated mutants is still inhibited, but to a lesser extent than in the wild type. Some of these mutants are higher candicidin producers than the wild type, not only in phosphate-supplemented medium but also in non-supplemented production medium. The high candicidin production by these mutants is due to (1) a high specific rate of candicidin biosynthesis and (2) an extended production phase. None of the phosphate-deregulated mutants in which uptake of [32P]phosphate was measured was a phosphate-permeability mutant.

Antifungal Agents↗