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Biomedical subjects

J S Feitelson

Publications and source records attributed to J S Feitelson.

9 recordsLinked to original sources

Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans.

The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in agriculture. Despite successful and extensive use of these toxins in transgenic crops, little is known about toxicity and resistance pathways in target insects since these organisms are not ideal for molecular genetic studies. To address this limitation and to investigate the potential use of these toxins to control parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans. We demonstrate for the first time that a single Bt toxin can target a nematode. When fed Bt toxin, C. elegans hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with toxin effects in insects. We have screened for and isolated 10 recessive mutants that resist the toxin's effects on the intestine, on fertility, and on viability. These mutants define five genes, indicating that more components are required for Bt toxicity than previously known. We find that a second, unrelated nematicidal Bt toxin may utilize a different toxicity pathway. Our data indicate that C. elegans can be used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold promise as nematicides.

Animals↗

Structure, function and engineering of Bacillus thuringiensis toxins.

Nature has provided potent insecticidal toxins as fermentation products of many Bacillus thuringiensis strains. Elucidation of structure-function relationships for this class of natural toxins is in its early stages. Both direct experimentation and application of theoretical structure-function principles emerging from the rapidly growing field of protein structure are accelerating understanding of these toxins. Coupled with the increasing demand for biologically sound pesticides, the benefits from engineering nature's toxins for improved performance set the stage for exciting, fast growth in discovery, characterization and commercialization of new active ingredients for biopesticides and transgenic plants.

Animals↗

New classes of Streptomyces coelicolor A3(2) mutants blocked in undecylprodigiosin (Red) biosynthesis.

Fifteen mutants of Streptomyces coelicolor A3(2) blocked in both the bipyrrole branch (redA) and a second site specific to the undecylprodigiosin pathway were characterized. Some of the mutants were ordered biosynthetically based on cosynthesis experiments. Complementation of each of the mutants with wild-type DNA cloned in low- and high-copy number plasmid vectors allowed the mutants to be separated into 12 new classes which are physically clustered within approximately 37 kb on the S. coelicolor genome. Early-step biosynthetic genes are centrally located and are flanked by later-step and regulatory genes.

DNA, Bacterial↗

Nucleotide sequence and transcriptional analysis of the redD locus of Streptomyces coelicolor A3(2).

Previous genetic evidence suggested that the redD gene product might be involved in the regulation of undecylprodigiosin (Red) biosynthesis in Streptomyces coelicolor. The redD+ gene was subcloned on a 2.2-kilobase-pair restriction fragment from the S. coelicolor redCD region by complementation of S. coelicolor JF1 (redD42). The DNA sequence of the 2.2-kilobase-pair redD-complementing region was determined, and the redD coding sequence was identified by computer analysis and deletion subcloning. Transcription at the redD locus was analyzed by using in vivo promoter probing, high resolution S1 mapping, and in vitro runoff transcription. A face-to-face arrangement of promoters was deduced, in which the proposed redD promoter was opposed by a cluster of four other promoters for another unidentified open reading frame. In time course experiments, redD transcription preceded that at two biosynthetic loci, redE and redBF; transcription at the latter two loci was reduced in redD42 mutants. The putative redD polypeptide lacked any strong sequence similarities to other known proteins.

Base Sequence↗

An improved plasmid for the isolation and analysis of Streptomyces promoters.

A Streptomyces promoter-probe plasmid vector was developed to isolate and characterize nucleotide sequences involved in transcriptional regulation. This pCLL34 vector was derived from plasmid pARC1 carrying the SLP1.2 origin of replication [Horinouchi and Beppu, J. Bacteriol. 162 (1985) 406-412]. Important features of the new vector include moderate host range, low copy number, simple identification of promoter activity by brown pigment production, an upstream transcriptional terminator, and a polylinker sequence containing a unique BamHI site for flexible cloning and fragment reisolation. The vector also provides the capability to directly determine the sequence of promoter-active inserts by dideoxy chain-termination sequencing methods using double-stranded plasmid templates and the appropriate oligodeoxynucleotide primers.

Base Sequence↗

Genetic and biochemical characterization of the red gene cluster of Streptomyces coelicolor A3(2).

Production of the red antibiotic, undecylprodigiosin, by Streptomyces coelicolor A3(2) was studied by DNA cloning and biochemical analysis. Over 21 kb of genomic DNA were cloned, in several segments, into plasmid vectors. The cloned DNA 'complemented' several specific mutations in the red gene cluster. Four red genes (redA, B, E, and F) were mapped to different regions within the cloned DNA. Screening with redE probes for DNA homologies among various streptomycetes revealed hybridizing DNA in three strains, one of them not known to synthesize prodigiosin pigments. Biochemical studies using protoplasted cells revised our interpretation of the nature of redE and redF mutations. Two forms of undecylnorprodigiosin: S-adenosylmethionine O-methyltransferase activity on gel filtration columns were detected: a very high molecular mass peak (greater than 5 MDal) and a 49 kDal) and a 49 kDal peak. Analyses of extracts from red mutants suggested that these two forms are related, and that at least the redE and redF gene products are necessary for O-methyltransferase activity in vivo. Lack of activity of the redE gene in a heterologous host, S. glaucescens, is consistent with the necessity for a biosynthetic complex involving several red gene products for efficient expression. Experiments in liquid antibiotic production medium indicated that prodigiosin compounds in S. coelicolor are examples of 'secondary metabolites' whose synthesis lags behind that of cell mass. The peak of specific activity of O-methyltransferase coincided with the 'late exponential' phase of growth. Thus, understanding the genetic regulation of undecylprodigiosin biosynthesis in S. coelicolor may be relevant to other antibiotic production pathways, and perhaps to 'secondary' metabolism in general.

Chromatography, Gel↗

Cloning of a Streptomyces gene for an O-methyltransferase involved in antibiotic biosynthesis.

The red pigmented antibiotic of Streptomyces coelicolor A3(2) is chemically very similar to the Serratia marcescens pigment, prodigiosin. We have demonstrated by co-synthesis experiments between non-producing mutants of both species that their biosynthetic pathways are similar, and have discovered identities between specific mutants of each organism. Molecular cloning techniques have been employed in order to isolate Streptomyces chromosomal DNA segments which "complement" a mutant defective in the penultimate step of the red biosynthetic pathway: an O-methyltransferase enzyme. In one case, the lesion appears to be repaired by integrative recombination into the chromosome; another case may represent expression from the autonomously replicating recombinant plasmid.

Anti-Bacterial Agents↗

Crude lysates of Staphylococcus aureus can transform Bacillus subtilis.

Plasmids can be transferred from Staphylococcus aureus to Bacillus subtilis by crude lysates prepared with penicillin or lysostaphin. These lysates mediate drug-resistance plasmid transformation in competent B. subtilis at an efficiency paralleling that of purified DNA.

Bacillus subtilis↗

Molecular genetics of Red biosynthesis in Streptomyces.

We have studied the molecular biology of undecylprodigiosin (Red) biosynthesis by Streptomyces coelicolor as a model system for understanding the genetic regulation of antibiotic biosynthesis. A collection of new red mutants was obtained using a directed screen. Regions of DNA involved in the transcriptional regulation of red gene expression were also isolated.

Base Sequence↗