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L C Vining

Publications and source records attributed to L C Vining.

At least 19 recordsLinked to original sources
PubMed1997

Probing the substrate specificity of an enzyme catalyzing inactivation of streptogramin B antibiotics using LC-MS and LC-MS/MS.

LC-MS and LC-MS/MS analyses indicated that an enzyme responsible for inactivating the antibiotic etamycin is specific for streptogramins and acts on both type B-I and B-II streptogramin subgroups. No enzymatic activity was detected for other cyclodepsipeptides such as surfactins and viscosin. It was demonstrated using analogs of etamycin that the picolinyl moiety is essential to obtain enzyme-generated ring-opened compounds. Because the picolinyl moiety is also essential for the biological activity of streptogramins, it is proposed that this residue is a distinctive topographic feature in the binding of this group of antibiotics to enzyme active sites.

Chromatography, Liquid↗
PubMed1997

Iterative type II polyketide synthases, cyclases and ketoreductases exhibit context-dependent behavior in the biosynthesis of linear and angular decapolyketides.

BACKGROUND: Iterative type II polyketide synthases (PKSs) produce polyketide chains of variable but defined length from a specific starter unit and a number of extender units. They also specify the initial regiospecific folding and cyclization pattern of nascent polyketides either through the action of a cyclase (CYC) subunit or through the combined action of site-specific ketoreductase (KR) and CYC subunits. Additional CYCs and other modifications may be necessary to produce linear aromatic polyketides. The principles of the assembly of the linear aromatic polyketides, several of which are medically important, are well understood, but it is not clear whether the assembly of the angular aromatic (angucyclic) polyketides follows the same rules. RESULTS: We performed an in vivo evaluation of the subunits of the PKS responsible for the production of the angucyclic polyketide jadomycin (jad), in comparison with their counterparts from the daunorubicin (dps) and tetracenomycin (tcm) PKSs which produce linear aromatic polyketides. No matter which minimal PKS was used to produce the initial polyketide chain, the JadD and DpsF CYCs produced the same two polyketides, in the same ratio; neither product was angularly fused. The set of jadABCED PKS plus putative jadl CYC genes behaved similarly. Furthermore, no angular polyketides were isolated when the entire set of jad PKS enzymes and Jadl or the jad minimal PKS, Jadl and the TcmN CYC were present. The DpsE KR was able to reduce decaketides but not octaketides; in contrast, the KRs from the jad PKS (JadE) or the actinorhodin PKS (ActIII) could reduce octaketide chains, giving three distinct products. CONCLUSIONS: It appears that the biosynthesis of angucyclic polyketides cannot be simply accomplished by expressing the known PKS subunits from artificial gene cassettes under the control of a non-native promoter. The characteristic structure of the angucycline ring system may arise from a kinked precursor during later cyclization reactions involving additional, but so far unknown, components of the extended decaketide PKS. Our results also suggest that some KRs have a minimal chain length requirement and that CYC enzymes may act aberrantly as first-ring aromatases that are unable to perform all of the sequential cyclization steps. Both of these characteristics may limit the widespread application of CYC or KR enzymes in the synthesis of novel polyketides.

Alcohol Oxidoreductases↗
PubMed1995

Inactivation of chloramphenicol by O-phosphorylation. A novel resistance mechanism in Streptomyces venezuelae ISP5230, a chloramphenicol producer.

Plasmid pJV4, containing a 2.4-kilobase pair insert of genomic DNA from the chloramphenicol (Cm) producer Streptomyces venezuelae ISP5230, confers resistance when introduced by transformation into the Cm-sensitive host Streptomyces lividans M252 (Mosher, R. H. Ranade, N. P., Schrempf, H., and Vining, L. C. (1990) J. Gen. Microbiol. 136, 293-301). Transformants rapidly metabolized Cm to one major product, which was isolated and purified by reversed phase chromatography. The metabolite was identified by nuclear magnetic resonance spectroscopy and mass spectrometry as 3'-O-phospho-Cm, and was shown to have negligible inhibitory activity against Cm-sensitive Micrococcus luteus. The nucleotide sequence of the S. venezuelae DNA insert in pJV4 contains an open reading frame (ORF) that encodes a polypeptide (19 kDa) with a consensus motif at its NH2 terminus corresponding to a nucleotide-binding amino acid sequence (motif A or P-loop; Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951). When a recombinant vector containing this ORF as a 1.6-kilobase pair SmaI-SmaI fragment was used to transform S. lividans M252, uniformly Cm-resistant transformants were obtained. A strain of S. lividans transformed by a vector in which the ORF had been disrupted by an internal deletion yielded clones that were unable to phosphorylate Cm, and exhibited normal susceptibility to the antibiotic. The results implicate the product of the ORF from S. venezuelae as an enzymic effector of Cm resistance in the producing organism by 3'-O-phosphorylation. We suggest the trivial name chloramphenicol 3'-O-phosphotransferase for the enzyme.

Amino Acid Sequence↗
PubMed1995

Chloramphenicol.

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Amino Acid Sequence↗
PubMed1995

Regulation of jadomycin B production in Streptomyces venezuelae ISP5230: involvement of a repressor gene, jadR2.

The nucleotide sequence of a region upstream of the type II polyketide synthase genes in the cluster for biosynthesis of the polyketide antibiotic jadomycin B in Streptomyces venezuelae contained an open reading frame encoding a sequence of 196 amino acids that resembeled sequences deduced for a group of repressor proteins. The strongest similarity was to EnvR of Escherichia coli, but the sequence also resembled MtrR, AcrR, TetC, and TcmR, all of which are involved in regulating resistance to antibiotics or toxic hydrophobic substances in the environment. Disruption of the nucleotide sequence of this putative S. venezuelae repressor gene (jadR2), by insertion of an apramycin resistance gene at an internal MluI site, and replacement of the chromosomal gene generated mutants that produced jadomycin B without the stress treatments (exposure to heat shock or to toxic concentrations of ethanol) required for jadomycin B production by the wild type. When cultures of the disruption mutants were ethanol stressed, they overproduced the antibiotic. From these results it was concluded that expression of the jadomycin B biosynthesis genes are negatively regulated by jadR2.

Amino Acid Sequence↗
PubMed1994

Cloning and sequence analysis of a recA-like gene from Streptomyces venezuelae ISP5230.

When genomic DNA fragments from Streptomyces venezuelae ISP5230 were probed at moderate stringency with recA from Mycobacterium tuberculosis, a 2.0-kb SmaI fragment was identified. The fragment was isolated by cloning a BamHI digest of S. venezuelae DNA in pHJL400 and screening the plasmids in Escherichia coli by Southern hybridization using a sib-selection technique. Sequencing the hybridizing region located an open reading frame encoding 377 amino acids. Its deduced amino acid sequence resembled that of recA genes from other bacteria. The cloned S. venezuelae gene conferred partial resistance to ethyl methanesulfonate when expressed in E. coli from the lacZ promoter.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of polyketide synthase genes for jadomycin B biosynthesis in Streptomyces venezuelae ISP5230.

Hybridizing fragments in the genomic DNA of Streptomyces venezuelae ISP5230, which produces the jadomycin group of angucycline antibiotics, were detected by probing with actI DNA from Streptomyces coelicolor A3(2). The hybridizing regions were isolated from a 16.5 kb insert of S. venezuelae DNA recovered from a genomic library cloned in a lambda replacement vector. Subcloning and sequencing of a 4.8 kb segment of the insert, containing regions hybridizing to actIII as well as actI, identified five open reading frames (ORFs). The deduced polypeptide products of the ORFs closely resemble in sequence the components of streptomycete type-II polyketide synthases (PKSs): the ORF1 product corresponds to the ketoacyl synthase, and the ORF2 product to a polypeptide closely related to the ketoacyl synthase and involved in determining chain length; the ORF3 product matches the acyl carrier protein; ORF4 encodes a bifunctional cyclase/dehydrase; and ORF5 encodes a ketoreductase. Integration into the chromosomal DNA of a plasmid containing a segment of the ORF2-ORF4 region severely depressed jadomycin B biosynthesis; since the integrant showed no change in growth or spore pigmentation, the cloned PKS genes are presumed to encode enzymes in the pathway for jadomycin biosynthesis.

Amino Acid Sequence↗
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↗