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L C Vining

Publications and source records attributed to L C Vining.

At least 37 records · Page 2Linked to original sources
PubMed1993

Organization of the genes encoding p-aminobenzoic acid synthetase from Streptomyces lividans 1326.

Genes involved in the biosynthesis of p-aminobenzoic acid (PABA) in Streptomyces lividans 1326 were cloned in pBR322 by complementing a pabB mutant of Escherichia coli. A 2.7-kb BamHI-SstI fragment of the cloned DNA complemented pabA and pabB mutations in both E. coli and S. lividans; complementation in S. lividans was accompanied by integration of the recombinant plasmid into the host chromosome. The nucleotide (nt) sequence of the 2.7-kb fragment contained two open reading frames, the deduced amino acid sequences of which were similar to those of pabA and pabB products from other bacteria. The nt sequences indicated that pabA and pabB are closely linked in S. lividans and supported cloning evidence that the genes are expressed from a promoter with features resembling those of most E. coli promoters.

Amino Acid Sequence↗
PubMed1993

Location of the genes for anthranilate synthase in Streptomyces venezuelae ISP5230: genetic mapping after integration of the cloned genes.

The anthranilate synthase (trpEG) genes in Streptomyces venezuelae ISP5230 were located by allowing a segregationally unstable plasmid carrying cloned S. venezuelae trpEG DNA and a thiostrepton resistance (tsr) marker to integrate into the chromosome. The integrated tsr was mapped by conjugation and transduction to a location close to tyr-2, between arg-6 and trpA13. A genomic DNA fragment containing trpC from S. venezuelae ISP5230 was cloned by complementation of a trpC mutation in Streptomyces lividans. Evidence from restriction enzyme analysis of the cloned DNA fragments, from Southern hybridization using the cloned trp DNA as probes, and from cotransduction frequencies, placed trpEG at a distance of 12-45 kb from the trpCBA cluster. The overall arrangement of tryptophan biosynthesis genes in the S. venezuelae chromosome differs from that in other bacteria examined so far.

Anthranilate Synthase↗
PubMed1993

Cloning, nucleotide sequence and expression in Streptomyces lividans and Escherichia coli of pabB from Lactococcus lactis subsp. lactis NCDO 496.

A gene (pabB) encoding the aminase activity of p-aminobenzoate (PABA) synthase in Lactococcus lactis subsp. lactis was cloned in pIJ41 and expressed in Streptomyces lividans strains defective in PABA biosynthesis. Expression of the gene was associated with a 1.2 kb deletion between the aph promoter and the cloning site in pIJ41. Subcloning in pBR322 and expression in Escherichia coli AB3295 of the cloned L. lactis DNA fragment localized the pabB-complementing gene in a 1.9 kb segment. The nucleotide sequence of this segment contained a 1410 bp open reading frame encoding a 470-amino-acid polypeptide of 50937 Da. The deduced amino acid sequence showed substantial similarity to those reported for PabB and TrpE from several organisms. Synonymous codon usage reflected the low G + C content in the genomic DNA of L. lactis subsp. lactis, and therefore differed markedly from the preferred usage in the S. lividans host. The cloned heterologous pabB DNA was expressed in amounts that allowed accumulation of excreted PABA in cultures of S. lividans transformants.

Amino Acid Sequence↗
PubMed1993

Accumulation of intracellular carbon reserves in relation to chloramphenicol biosynthesis by Streptomyces venezuelae.

Two chloramphenicol-producing strains of Streptomyces venezuelae accumulated small amounts of polyhydroxybutyrate during exponential growth; the compound disappeared from the mycelium as the cultures entered stationary phase. Depletion of polyhydroxybutyrate coincided with chloramphenicol production but the amount of polymer stored in the mycelium was insufficient to supply the precursor requirement for biosynthesis of the antibiotic. Accumulation of polyhydroxybutyrate in the S. venezuelae strains was appreciably lower than in two other streptomycetes examined. Glycogen and lipids accumulated in the mycelium of S. venezuelae 13s during the stationary phase, after nitrogen depletion; under the culture conditions used, they were the principal storage compounds in S. venezuelae. Trehalose was absent from the mycelium in vegetative cultures grown under nonsporulating conditions but it was abundant in spores obtained from submerged and surface cultures. Glycogen and polyhydroxybutyrate were absent from spores.

Chloramphenicol↗
PubMed1993

Nutrient effects on growth and the production of 5-hydroxy-4-oxonorvaline by Streptomyces akiyoshiensis.

5-Hydroxy-4-oxonorvaline (HON) was produced optimally by Streptomyces akiyoshiensis in media containing starch and casein supplemented with high concentrations of phosphate and magnesium sulfate; cultures grown at initial pH values between 6.3 and 6.6 yielded HON titres of 13-14 mM. Physiological analysis of HON production provided evidence that the excess inorganic constituents in this medium played an important role in optimizing production. In simple defined media buffered at pH 5.5-6.0 and inoculated with either a spore suspension or washed vegetative mycelium, formation of HON was less than one-third the amount produced in the starch-casein-salts medium. Production was markedly affected by the carbon and nitrogen sources used; media containing starch and potassium nitrate or aspartate supported the highest yields of HON. In starch-nitrate media, production was suppressed by excess nitrogen source, and the HON titre decreased 3-fold as the phosphate content was increased from 0.5 to 5 mM; growth was not appreciably altered. The results suggest that the high level of HON production in the complex starch-casein-salts medium is associated with sequestration of metabolic ammonium and reduced availability of phosphate through formation of the poorly soluble magnesium ammonium phosphate.

Aminolevulinic Acid↗
PubMed1992

Secondary metabolism, inventive evolution and biochemical diversity--a review.

Evidence now being obtained through nucleotide (nt) sequence analysis supports the concept that secondary metabolism has arisen by modification of existing primary metabolic reactions. Although amino acid sequence identity deduced from nt sequences of genes encoding proteins from related primary and secondary metabolic pathways is sufficient to indicate a common ancestry, the match is often better when genes in different rather than in the same species are compared. The information so far available suggests that gene transfer between organisms has been an important factor in the evolution of secondary metabolism. Many secondary pathways may be of relatively ancient origin and they may have arisen only infrequently. Much subsequent elaboration of the pathways has probably taken place after their acquisition by other species and so has been influenced by a variety of selective conditions. The characteristic diversity of secondary metabolites and their functions can be accounted for by the random manner in which the pathways initially evolved and have subsequently been exploited.

Anti-Bacterial Agents↗
PubMed1992

Roles of secondary metabolites from microbes.

The common feature of the seemingly diverse array of biological activities exhibited by microbial secondary metabolites is their survival value for the producing organism. The propensity to form these compounds is unevenly distributed in microbial taxa and seems more closely associated with existence in a competitive environment than with phylogeny. The characteristic multibranched elaboration of secondary biosynthetic pathways and the marked species specificity of the end products are consistent with their evolution by an 'inventive' mechanism. The species specificity suggests that distinctive terminal reactions may be of recent origin. However, comparisons of the nucleotide sequence of genes involved in the biosynthesis of phenazine and polyketide metabolites with related genes of primary pathways indicate that the secondary pathways have not evolved exclusively within the organisms in which they are now found. Sequence similarities with related primary pathway genes in phylogenetically distant organisms suggest that gene transfer has played an important part in the evolution of secondary metabolism. The diversity of products may reflect the many roles for which secondary metabolites have been selected after the genes for their biosynthesis have transferred to organisms with different physiologies and different environment challenges.

Bacteria↗
PubMed1992

Growth morphology of Streptomyces akiyoshiensis in submerged culture: influence of pH, inoculum, and nutrients.

Most media in which the growth of shaken submerged cultures of Streptomyces akiyoshiensis was examined did not support the formation of well-dispersed mycelial suspensions. Investigation of the culture conditions promoting dispersed growth showed the pH of the culture medium to be of critical importance; an initial value of 5.5 minimized aggregation of the mycelium while supporting adequate biomass production. In cultures started at this pH, spore inocula gave better mycelial dispersal than did vegetative inocula; with spore inocula, growth morphology was also less affected by inoculum size. The composition of the nutrient solution influenced the extent of mycelial dispersal; slow growth was often associated with clumping but no clear correlation was observed between pellet formation and the ability of carbon or nitrogen sources to support rapid growth. Increasing the phosphate concentration from 0.5 to 15 mM caused a modest decrease in mycelial aggregation. Conditions promoting a well-dispersed mycelium suitable for studying the physiological control of secondary metabolism also supported the formation of 5-hydroxy-4-oxonorvaline by S. akiyoshiensis.

Aminolevulinic Acid↗
PubMed1991

Molecular cloning of the genes for anthranilate synthetase from Streptomyces venezuelae ISP 5230.

Fragments of genomic DNA from Streptomyces venezuelae ISP5230 were cloned in the Escherichia coli expression vector pTZ18R and the plasmids were used to transform E. coli JA194 (trpE). The transformants included a prototrophic strain containing a recombinant plasmid, pDQ181, with an approximately 6.8-kb insert. Subcloning located the trpE-complementing DNA in a 2.4-kb segment. Transformation of E. coli ED23 (lacking both trpE and trpG functions) with plasmids containing the 2.4-kb DNA segment gave prototrophic strains exhibiting both the ASI and ASII activities of anthranilate synthetase. The results indicated that trpE and trpG are clustered in S. venezuelae. Regions hybridizing to the pDQ181 insert were present in the genomic DNA of other streptomycetes.

Anthranilate Synthase↗
PubMed1991

Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli.

Lysine epsilon-aminotransferase (LAT) in the beta-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. Cloning of restriction fragments from Streptomyces clavuligerus, a beta-lactam producer, into Streptomyces lividans, a nonproducer that lacks LAT activity, led to the production of LAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a single open reading frame encoding a polypeptide with an approximately Mr 49,000. Expression of this coding sequence in Escherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived from a single polypeptide. A second open reading frame began immediately downstream from lat. Comparison of this partial sequence with the sequences of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases from Penicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gramicidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of the putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies approximately 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the beta-lactam biosynthetic cluster between pcbC and cefE (approximately 25 kbp) is nearly complete.

Amino Acid Sequence↗
PubMed1991

Cloning and location of a gene governing lysine epsilon-aminotransferase, an enzyme initiating beta-lactam biosynthesis in Streptomyces spp.

In actinomycetes that produce beta-lactam antibiotics of the cephem type, lysine epsilon-aminotransferase is the initial enzyme in the conversion of lysine to alpha-aminoadipic acid. We used a two-stage process ("chromosome walking") to screen a lambda library of Streptomyces clavuligerus genomic DNA for fragments that expressed lysine epsilon-aminotransferase activity in S. lividans. Restriction analysis of the cloned DNA confirmed the location of the putative lat gene within the cluster of beta-lactam biosynthesis genes, roughly midway between pcbC, the structural gene for isopenicillin N synthetase, and the putative cefE gene encoding deacetoxycephalosporin C synthetase.

Anti-Bacterial Agents↗
PubMed1990

Nutritional control of actinorhodin production by Streptomyces coelicolor A3(2): suppressive effects of nitrogen and phosphate.

Actinorhodin production in Streptomyces coelicolor A3(2) was relatively insensitive to the carbon source concentration but was elicited by nitrogen or phosphate depletion, or by a decline in the growth rate. In starch-glutamate media with nitrogen limitation, increasing the nitrogen supply delayed the onset of antibiotic synthesis and, at concentrations above 30 mM, decreased its rate. In a similar medium with phosphate limitation, increasing the initial phosphate concentration delayed actinorhodin formation and, above 2.5 mM, reduced the rate of synthesis. Experiments in which actinorhodin synthesis was elicited by phosphate depletion at various nitrogen concentrations demonstrated strong suppression by residual glutamate. Cultures in which actinorhodin biosynthesis was initiated by nitrogen depletion were not similarly suppressed by increasing amounts of residual phosphate. The results suggest that actinorhodin production in S. coelicolor A3(2) responds to interacting physiological controls, notable among which is nitrogen catabolite regulation.

Anthraquinones↗
PubMed1990

Physiology of antibiotic production in actinomycetes and some underlying control mechanisms.

Some of the accumulated information on the physiology and nutritional control of antibiotic production in actinomycetes can now be related to recent discoveries in the field of actinomycete molecular biology. This review focuses on aspects of genetic and metabolic control of antibiotic biosynthesis. It surveys some well established principles in the relationship between primary and secondary metabolism, and summarizes briefly the areas where progress is being made in elucidating the molecular organization of regulatory systems underlying this relationship.

Journal Article↗
PubMed1990

Biosynthesis and control of beta-lactam antibiotics: the early steps in the "classical" tripeptide pathway.

The interaction between growth and secondary metabolism develops from physiological responses of the producer organism to its environment. Nutrients are channelled into primary growth processes or into secondary processes such as antibiotic biosynthesis by a variety of metabolic controls, the nature of which has been extensively studied in organisms producing beta-lactam antibiotics via the tripeptide, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine. In the following article we review the early stages of beta-lactam biosynthesis in fungi and actinomycetes, keeping in mind the regulation of primary pathways that provide the amino acid precursors of this group of antibiotics, as well as the regulation of the secondary pathway itself. Of special importance to organisms engaging in secondary metabolism are the control mechanisms that suppress the nonessential process during rapid growth but allow secondary metabolic genes to be expressed and resources to be diverted when environmental factors generate the appropriate biochemical signals.

Journal Article↗
PubMed1990

Chloramphenicol resistance in Streptomyces: cloning and characterization of a chloramphenicol hydrolase gene from Streptomyces venezuelae.

A 6.5 kb DNA fragment containing a chloramphenicol-resistance gene of Streptomyces venezuelae ISP5230 was cloned in Streptomyces lividans M252 using the high-copy-number plasmid vector pIJ702. The gene was located within a 2.4 kb KpnI-SstI fragment of the cloned DNA and encoded an enzyme (chloramphenicol hydrolase) that catalysed removal of the dichloroacetyl moiety from the antibiotic. The deacylated product, p-nitrophenylserinol, was metabolized to p-nitrobenzyl alcohol and other compounds by enzymes present in S. lividans M252. Examination of the genomic DNA from several sources using the cloned 6.5 kb SstI fragment from S. venezuelae ISP5230 as a probe showed a hybridizing region in the DNA from S. venezuelae 13s but none in the DNA from another chloramphenicol producer, Streptomyces phaeochromogenes NRRLB 3559. The resistance phenotype was not expressed when the 6.5 kb SstI fragment or a subfragment was subcloned behind the lac-promoter of plasmid pTZ18R in Escherichia coli.

Amidohydrolases↗
PubMed1990

Sporulation of Streptomyces venezuelae in submerged cultures.

Shaken cultures of Streptomyces venezuelae ISP5230 in minimal medium with galactose and ammonium sulphate as carbon and nitrogen sources, respectively, showed extensive sporulation after 72 h incubation at 37 degrees C. The spores formed in these cultures resembled aerial spores in their characteristics. The ability of the spores to withstand lysozyme treatment was used to monitor the progress of sporulation in cultures and to determine the physiological requirements for sporulation. In media containing ammonium sulphate as the nitrogen source, galactose was the best of six carbon sources tested. With galactose S. venezuelae ISP5230 sporulated when supplied with any of several nitrogen sources; however, an excess of nitrogen source was inhibitory. In cultures containing galactose and ammonium sulphate, sporulation was suppressed by a peptone supplement. The onset of sporulation was accompanied by a drop in intracellular GTP content. When decoyinine, an inhibitor of GMP synthase, was added to a medium containing starch and ammonium sulphate, a slight increase in sporulation was seen after 2 d. The suppression of sporulation by peptone in liquid or agar cultures was not reversed by addition of decoyinine. A hypersporulating mutant of S. venezuelae ISP5230 was altered in its ability to assimilate sugars. In cultures containing glucose the mutant sporulated more profusely than did the wild-type and did not acidify the medium to the same extent. However, the suppressive effect of glucose on sporulation was not merely a secondary result of acid accumulation.

Adenosine↗
PubMed1990

Plasmid transformation of Streptomyces venezuelae: modified procedures used to introduce the gene(s) for p-aminobenzoate synthase.

Sucrose was unsuitable as an osmotic stabilizer in buffer solutions and media used for transformation of Streptomyces venezuelae ISP5230. Its replacement with NaCl, together with other modifications in the procedure, allowed efficient formation and regeneration of protoplasts but did not support transformation of S. venezuelae ISP5230 by vectors pIJ41 and pIJ941. With pIJ702, transformants with a low plasmid-copy-number and altered growth characteristics were obtained. Both pIJ702 and pIJ941, but not pIJ41, transformed S. venezuelae 13s; when pIJ941 was used, the plasmid in 18 of 20 transformants contained a deletion in the region reported to code for replication and transfer. The modified plasmid transformed S. venezuelae ISP5230 efficiently and was used to introduce a fragment of DNA from the pab locus of the wild-type into a Cml-1 mutant of ISP5230 blocked in chloramphenicol formation. Transformants that overproduced p-aminobenzoic acid were obtained but they remained blocked in chloramphenicol production; thus, the cloned pab fragment did not contain genes able to complement the cml-1 mutation. The results also suggest that the Cml-1 phenotype is not due to a defective reaction common to the biosynthesis of p-aminobenzoic acid and chloramphenicol.

4-Aminobenzoic Acid↗