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Biomedical subjects

A L Demain

Publications and source records attributed to A L Demain.

At least 55 records · Page 3Linked to original sources
PubMed1996

Unexpected enhancement of beta-lactam antibiotic formation in Streptomyces clavuligerus by very high concentrations of exogenous lysine.

L-Lysine is known to stimulate production of beta-lactam antibiotics by Streptomyces clavuligerus via provision of the lysine breakdown product, L-alpha-aminoadipic acid, which is a limiting precursor. Previous investigations utilized levels of 10-20 mM L-lysine as an addition to chemically-defined media resulting in 50-100% improvement in antibiotic production. We were surprised to note that as the concentration was further increased, the organism responded by producing even higher titers of antibiotics. The optimum concentration of 100 mM L-lysine yielded an approximate 500% increase in production with only minor effects on growth. DL- and D-Lysine also exerted enhancements suggesting the presence of a lysine racemase or some other route from D-lysine to L-alpha-aminoadipate in this organism; D-lysine was considerably less potent than DL- or L-lysine.

Anti-Bacterial Agents↗
PubMed1996

Interactions between gramicidin S and its producer, Bacillus brevis.

Gramicidin S (GS) inhibition of germination outgrowth of Bacillus brevis spores was reversed completely by a short pretreatment with sodium dodecyl sulfate, moderately by ethanol or by incubation at pH 10 but not by incubation at pH 4. Of five metal ions tested (Na+, Mg2+, Fe2+, Cu2+, Ca2+), only Ca2+ reversed GS inhibition. When Ca2+ (but not the other four metal ions) was added to the growth medium, there was a considerable portion of the biosynthesized GS found in the extracellular fluid. These findings are interpreted in terms of the binding of GS to the external layers of the B. brevis spore.

Anti-Bacterial Agents↗
PubMed1996

Restriction endonuclease activity in Clostridium thermocellum and Clostridium thermosaccharolyticum.

Clostridium thermocellum cell extracts exhibit specific endonuclease activity with very little non-specific exonuclease activity at 55 degrees C. The Dam methylation system of Escherichia coli offers complete protection from digestion by C. thermocellum ATCC 27405 cell extracts for all DNA tested (totaling > 100 kb, insuring that most potential restriction sequences have been exposed). Based on both the Dam recognition sequence and the similarity of cell extract and MboI DNA digests, the C. thermocellum restriction enzyme recognition sequence appears to be 5' GATC 3'. Cell extracts made from a second thermophile, C. thermosaccharolyticum ATCC 31960 do not exhibit specific endonuclease activity under the conditions tested. Genomic DNA from C. thermocellum exhibits a Dam+ phenotype while genomic DNA from C. thermosaccharolyticum exhibits a Dam- phenotype.

Biotechnology↗
PubMed1996

FACS-based isolation of slowly growing cells: double encapsulation of yeast in gel microdrops.

Isolating hyperproducing cells is important in biotechnology, but these cells usually grow slowly and can be overgrown by poorly producing cells. We describe a new method of isolating slowly growing cells from among rapidly growing cells, which has the potential for automation and high throughput (e.g., 100,000 cells/h). A model system is presented consisting of a mixed population of slowly growing mutant and rapidly growing wild-type yeast, which were encapsulated in double agarose gel microdrops (dGMDs); with most dGMDs initially containing single cells. Double encapsulation locates parent cells near dGMD centers, making microcolony measurement more accurate. After a 15-h incubation, fluorescent activated cell sorting was used to analyze and sort dGMDs with small microcolonies (slow growers) from dGMDs with large microcolonies (rapid growers). Successful isolation of slow growers from a mixed population of predominantly rapidly growing Saccharomyces cerevisiae cells was achieved.

Cell Division↗
PubMed1996

Effect of water activity on production of beta-lactam antibiotics by Streptomyces clavuligerus in submerged culture.

The amount of available water in the environment of micro-organisms, defined as water activity (aw), has been shown to affect growth, respiration, enzyme synthesis, sporulation and other physiological functions. The aim of this study was to examine the impact of aw on production/excretion of a secondary metabolite. For this purpose, the production of beta-lactam antibiotics and biomass of Streptomyces clavuligerus was studied in relation to the aw-depressing agents glucose, sorbitol and NaCl. These were chosen because NaCl and sorbitol are often used to depress aw and glucose was not thought to be taken up by S. clavuligerus. The filamentous bacterium S. clavuligerus NRRL 3585 (ATCC 27064) is a prokaryotic producer of penicillin N, cephalosporins including cephamycin C and clavulanic acid. Under water stress conditions, a greater effect upon antibiotic biosynthesis than upon growth was consistently observed. When aw was decreased to below 0.997, antibiotic production began to decrease. For growth, inhibition was much more gradual and did not become intensive until an aw of 0.990 was reached.

Anti-Bacterial Agents↗
PubMed1995

The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome.

Enzymatic cellulose degradation is a heterogeneous reaction requiring binding of soluble cellulase molecules to the solid substrate. Based on our studies of the cellulase complex of Clostridium thermocellum (the cellulosome), we have previously proposed that such binding can be brought about by a special "anchorage subunit." In this "anchor-enzyme" model, CipA (a major subunit of the cellulosome) enhances the activity of CelS (the most abundant catalytic subunit of the cellulosome) by anchoring it to the cellulose surface. We have subsequently reported that CelS contains a conserved duplicated sequence at its C terminus and that CipA contains nine repeated sequences with a cellulose binding domain (CBD) in between the second and third repeats. In this work, we reexamined the anchor-enzyme mechanism by using recombinant CelS (rCelS) and various CipA domains, CBD, R3 (the repeat next to CBD), and CBD/R3, expressed in Escherichia coli. As analyzed by non-denaturing gel electrophoresis, rCelS, through its conserved duplicated sequence, formed a stable complex with R3 or CBD/R3 but not with CBD. Although R3 or CBD alone did not affect the binding of rCelS to cellulose, such binding was dependent on CBD/R3, indicating the anchorage role of CBD/R3. Such anchorage apparently increased the rCelS activity toward crystalline cellulose. These results substantiate the proposed anchor-enzyme model and the expected roles of individual CipA domains and the conserved duplicated sequence of CelS.

Bacterial Proteins↗
PubMed1995

Effect of amino acids on rapamycin biosynthesis by Streptomyces hygroscopicus.

In a chemically defined medium containing aspartate, arginie and histidine to support good growth, addition of L-lysine stimulated rapamycin production by 150%. This was probably due to its conversion to pipecolic acid, a rapamycin precursor. Phenylalanine and methionine interfered in rapamycin production by unknown mechanisms.

Amino Acids↗
PubMed1995

Phosphate, ammonium, magnesium and iron nutrition of Streptomyces hygroscopicus with respect to rapamycin biosynthesis.

Phosphate, ammonium and magnesium salts interfered with rapamycin production by Streptomyces hygroscopicus at concentrations optimal for growth. These observations point to the existence of phosphorus, magnesium and nitrogen-negative regulation mechanisms for rapamycin biosynthesis. On the other hand, Fe2+ stimulated rapamycin production at concentrations greater than that required for growth.

Antifungal Agents↗
PubMed1995

Dependence of nitrogen- and phosphorus-regulation of beta-lactam antibiotic production by Streptomyces clavuligerus on aeration level.

Interference with beta-lactam production in Streptomyces clavuligerus by ammonium and phosphate ions, normally observed with optimum levels of aeration, was eliminated by restriction of the air supply. Under such a restriction, ammonium slightly stimulated and phosphate markedly stimulated beta-lactam antibiotic production. These are rare examples of 'regulation reversal' by an environmental modification.

Air↗
PubMed1995

Carbon source nutrition of rapamycin biosynthesis in Streptomyces hygroscopicus.

Chemically-defined media were developed for rapamycin production by Streptomyces hygroscopicus. Thirty-five carbon sources were tested for their effect on production. Eight failed to support growth and seven appeared to repress or inhibit rapamycin formation. The best combination of two carbon sources were 2% fructose and 0.5% mannose. Acetate and propionate, which are known to contribute most of the carbon atoms of the lactone ring, were unsatisfactory for growth and/or rapamycin production.

Antibiotics, Antineoplastic↗
PubMed1995

Exogenous shikimic acid stimulates rapamycin biosynthesis in Streptomyces hygroscopicus.

A chemically defined medium was developed which satisfies the nutritional needs of the Streptomyces hygroscopicus strain producing the immunosuppressant rapamycin. More than a doubling of rapamycin biosynthesis was observed on addition of 57 mmol/L of exogenous shikimate. The previously observed inhibition of the synthesis by phenylalanine was not reversed by shikimate.

Immunosuppressive Agents↗
PubMed1995

Beta-lactams.

Explore the source record for details and available documents.

Acremonium↗
PubMed1994

Interdependence of gene expression for early steps of cephalosporin synthesis in Streptomyces clavuligerus.

The early steps of cephamycin synthesis by S. clavuligerus are catalyzed sequentially by lysine epsilon-aminotransferase (LAT), delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (cyclase, IPNS). The genes (lat, pcbAB, and pcbC, respectively) are closely linked in the same order as the enzymes act in the biosynthetic pathway and are transcribed in the same direction. Four cephamycin non- (or low-) producing mutants are pleiotropic in that they have undetectable or markedly diminished levels of ACVS and cyclase; two mutants almost completely lack LAT activity. All four mutants are complemented in cephamycin formation by transformation with pNBR1, a plasmid containing a 7.2-kb genomic region of S. clavuligerus in vector pIJ702. The cloned DNA was found to possess no part of the cyclase gene, but instead it contained lat and the 5' upstream part of pcbAB. Doran et al. reported that the 31-bp region between pcbAB and pcbC contains no recognizable promoter or transcription termination sequences. We found that there are 153 bp between the lat ORF and the pcbAB start codon. A potential transcriptional terminator begins 4 to 6 bp downstream of the lat ORF. In the 111-bp segment between the end of the "terminator" and the pcbAB start codon, there are no Streptomyces-like or Escherichia coli-like promoter consensus sequences. However, upstream of the "terminator," that is, in the downstream portion of the lat ORF, are two regions resembling a Streptomyces consensus promoter. Promoter activity in gene fusion constructions was demonstrated in this region. A third potential promoter is upstream of the lat ORF, but only the--10 part is on the cloned DNA. The mechanism by which the cloned DNA (containing lat, the 5' part of pcbAB, and the intervening sequence) influences the expression of the downstream genes encoding ACVS and IPNS, even in strains that possess LAT activity, is an intriguing target of future investigation.

Cephalosporins↗
PubMed1994

A role for alanine in the ammonium regulation of cephalosporin biosynthesis in Streptomyces clavuligerus.

It is known that excess ammonium supply decreases cephalosporin production and represses cephalosporin synthases. We wondered whether an additional important effect could be inhibition of synthase action by alanine. We had previously shown that ammonium addition induced alanine dehydrogenase and increased intracellular alanine and that alanine could inhibit resting cell synthesis of cephalosporins. In the present work we confirm the alanine inhibition of antibiotic production by resting cells. We found L-alanine inhibited three of the four synthases tested: ACV synthetase, cyclase and expandase; the epimerase was not inhibited. These data suggest that interference in cephalosporin production by growth in ammonium salts involves synthase inhibition by intracellular alanine, in addition to the known role of ammonium in synthase repression.

Alanine↗
PubMed1994

Possible involvement of the lysine epsilon-aminotransferase gene (lat) in the expression of the genes encoding ACV synthetase (pcbAB) and isopenicillin N synthase (pcbC) in Streptomyces clavuligerus.

Streptomyces clavuligerus produces the beta-lactam antibiotics penicillin N, O-carbamoyldeacetylcephalosporin C and cephamycin C. We characterized a wild-type DNA region which restores antibiotic formation to a mutant strain named NP1, previously shown to exhibit depressed activities for two early enzymes of cephalosporin synthesis, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (IPNS). L-Lysine epsilon-aminotransferase (LAT) assays and alpha-AAA feeding experiments suggested that strain NP1 is a lat mutant. NP1 recovered LAT, ACVS and IPNS activities when transformed with the cloned region. DNA sequencing showed that this region encodes the entire LAT gene (lat), required for the conversion of L-lysine to the beta-lactam precursor L-alpha-aminoadipic acid (alpha-AAA), as well as the upstream half of the ACVS gene (pcbAB). The activities of ACVS and IPNS appear to depend upon LAT expression. Gene fusions constructed to investigate promoter activities in the cloned region support a model of interdependence in the expression of the genes for LAT, ACVS and IPNS (pcbC).

Amino Acid Sequence↗
PubMed1993

Purification and properties of the Clostridium thermocellum bglB gene product expressed in Escherichia coli.

The Clostridium thermocellum beta-glucosidase B was purified to homogeneity in its recombinant form from Escherichia coli. The purification protocol included ion exchange, hydrophobic interaction and hydroxyapatite chromatography. The polypeptide was found to have a molecular mass of 84,000 daltons and a pI of 4.4. There was a differential effect of temperature on the aryl-beta-glucosidase and cellobiase activities of the purified protein. The cellobiase activity had an optimum of 45 degrees C, and aryl-beta-glucosidase 60 degrees C. Both activities had an optimum pH of 5.6, although the aryl-beta-glucosidase had a secondary peak at 7.0. Both activities were stimulated by divalent cations and DTT, but inhibited by thiol reagents. The enzyme was found to have a broad substrate specificity. Using cellobiose as substrate and a temperature of 45 degrees C, the Km and Vmax values were 1.6 mM and 5.5 U mg-1 respectively. The aryl-beta-glucosidase when assayed against pNP glucopyranoside and a temperature of 60 degrees C had Km and Vmax of 2.9 mM and 1.1 U mg-1 respectively. The enzyme was very stable at 45 degrees C, but rapidly inactivated at 60 degrees C.

Carbohydrate Sequence↗