Influence of ancillary genes, encoding aspects of methionine metabolism, on tylosin biosynthesis in Streptomyces fradiae.

The tylosin-biosynthetic (tyl) gene cluster of Streptomyces fradiae contains ancillary genes that encode functions normally associated with primary metabolism. These can be disrupted without loss of viability, since equivalent genes (presumably used for 'housekeeping' purposes) are also present elsewhere in the genome. The tyl cluster also contains two genes that encode products unlike any proteins in the databases. Two ancillary genes, metF (encoding N5,N10-methylenetetrahydrofolate reductase) and metK, encoding S-adenosylmethionine synthase, flank one of the 'unknown' genes (orf9) in the tyl cluster. In a strain of S. fradiae in which all three of these genes were disrupted, tylosin production was reduced, although this effect was obscured in media supplemented with glycine betaine which can donate methyl groups to the tetrahydrofolate pool. Apparently, one consequence of the recruitment of ancillary genes into the tyl cluster is enhanced capacity for transmethylation during secondary metabolism.

The tylosin-biosynthetic genes of Streptomyces fradiae.

The tylosin-biosynthetic (tyl) gene cluster occupies about 1% of the genome of Streptomycesfradiae and includes at least 43 open reading frames. In addition to structural genes required for tylosin production, the tyl cluster contains three resistance determinants and several regulatory genes. Tylosin production is evidently controlled by pathway-specific and pleiotropic regulators with the likely involvement of y-butyrolactone signalling factors. Accumulation of the polyketide aglycone is controlled by glycosylated macrolides and optimal performance of the complex polyketide synthase enzyme requires the activity of an editing thioesterase.

Multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae.

BACKGROUND: The macrolide antibiotic tylosin is composed of a polyketide lactone substituted with three deoxyhexose sugars. In order to produce tylosin efficiently, Streptomyces fradiae presumably requires control mechanisms that balance the yields of the constituent metabolic pathways together with switches that allow for temporal regulation of antibiotic production. In addition to possible metabolic feedback and/or other signalling devices, such control probably involves interplay between specific regulatory proteins. Prior to the present work, however, no candidate regulatory gene(s) had been identified in S. fradiae. RESULTS: DNA sequencing has shown that the tylosin biosynthetic gene cluster, within which four open reading frames utilise the rare TTA codon, contains at least five candidate regulatory genes, one of which (tylP) encodes a gamma-butyrolactone signal receptor for which tylQ is a probable target. Two other genes (tylS and tylT) encode pathway-specific regulatory proteins of the Streptomyces antibiotic regulatory protein (SARP) family and a fifth, tylR, has been shown by mutational analysis to control various aspects of tylosin production. CONCLUSIONS: The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.

Analysis of four tylosin biosynthetic genes from the tylLM region of the Streptomyces fradiae genome.

The tylLM region of the tylosin biosynthetic gene cluster of Streptomyces fradiae contains four open reading frames (orfs1*-4*). The function of the orf1* product is not known. The product of orf2* (tylM2) is the glycosyltransferase that adds mycaminose to the 5-hydroxyl group of tylactone, the polyketide aglycone of tylosin (Ty). A methyltransferase, responsible for 3-N-methylation during mycaminose production, is encoded by orf3* (tylM1). The product of orf4* (cer) is crotonyl-CoA reductase, which converts acetoacetyl-CoA to butyryl-CoA for use as a 4C extender unit during tylactone production.

Molecular analysis of tlrD, an MLS resistance determinant from the tylosin producer, Streptomyces fradiae.

The macrolide antibiotic, tylosin (Ty), is produced by Streptomyces fradiae. Two resistance determinants (tlrA, synonym ermSF, and tlrD) conferring resistance to macrolide, lincosamide and streptogramin B type (MLS) antibiotics were previously isolated from this strain, and their products shown to methylate 23S ribosomal RNA (rRNA) at a common site, thereby rendering the ribosomes MLS resistant. However, the TlrA and TlrD proteins differ in their action; the former dimethylates, and the latter monomethylates, the target nucleotide. Here, 2.2 kb of DNA from the tylLM region of the tylosin biosynthetic gene cluster of S. fradiae has been sequenced and shown to encompass tlrD. Comparison of the sequences of tlrA and tlrD (and of their deduced products) with those of related ('erm-type') genes from other actinomycetes suggests that the combined presence of tlrA and tlrD in S. fradiae is not the result of recent gene duplication.

Antigen detection using recombinant, bifunctional single-chain Fv fusion proteins synthesised in Escherichia coli.

A gene fusion approach has been used to produce antibody conjugates for use in immunoassays. Escherichia coli expression vectors encoding fusions between the outer membrane protein A signal peptide, an anti-phytochrome single-chain Fv protein, and either Escherichia coli alkaline phosphatase or Staphylococcal protein A downstream of the T7 O10 promoter were constructed. A crude lysate from cells expressing the single-chain Fv-alkaline phosphatase fusion protein could be used directly for the sensitive and specific staining of phytochrome on protein blots by a single-step immunoassay procedure. Following purification by immunoglobulin G affinity chromatography, the Staphylococcal protein A-single-chain Fv fusion protein was also used for selective immunostaining of phytochrome on protein blots by a two-step procedure in which a rabbit immunoglobulin-alkaline phosphatase conjugate was used to detect antigen-bound Staphylococcal protein A. Recombinant antibody conjugates of the types described here are simple and inexpensive to produce and are a realistic alternative to conventional antibody conjugates.

Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures.

A synthetic gene encoding an anti-phytochrome single-chain Fv (scFv) antibody bearing an N-terminal signal peptide has been used to transform tobacco plants. Immunoblot analysis showed that transformed plants accumulate high levels of scFv protein, accounting for up to 0.5% of the total soluble protein fraction, which could be extracted by simple infiltration and centrifugation of leaf tissue. A substantial proportion of the scFv protein extracted in this way was found to possess antigen-binding activity. Callus cell suspension cultures derived from transformed plants secrete functional scFv protein into the surrounding medium. Compared with the levels of scFv protein observed in plants expressing the native scFv gene, the incorporation of an N-terminal signal peptide, to target the scFv to the apoplast, results in elevated accumulation of the protein.

Production and secretion of a bifunctional staphylococcal protein A::antiphytochrome single-chain Fv fusion protein in Escherichia coli.

A bifunctional molecule was genetically engineered which contained the secretory signal and four Fc-binding domains of Staphylococcus aureus protein A (FcA), fused to a single-chain Fv (scFv) derived from an immunoglobulin (Ig) G1 mouse monoclonal antibody (AS32) directed against the plant regulatory photoreceptor protein, phytochrome. The FcA::AS32scFv sequence was encoded in a single synthetic gene and expressed as a 60-kDa periplasmic protein in Escherichia coli. The bifunctionality of the fusion protein was established by its ability to bind to both IgG-agarose and phytochrome-sepharose. Growth of cultures, producing the FcA::AS32scFv, at 37 degrees C, resulted in a decrease in the periplasmic accumulation of the fusion protein, and an increased accumulation of an assumed degradation product which retained Fc-binding activity. Growth of cultures at lower temperatures favoured the accumulation of undegraded fusion protein. The recombinant fusion protein could be purified to homogeneity by a simple, rapid chromatography procedure.

Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco.

We have expressed a synthetic gene that encodes an antigen-binding single-chain FV protein (scFV) in transgenic tobacco plants. The scFV gene was created by polymerase chain reaction (PCR) amplification of the variable domain coding regions from a mouse monoclonal hybridoma cell line. The monoclonal cell line secretes an IgG1 antibody that binds to the plant regulatory photoreceptor protein, phytochrome. The cloned scFV gene was expressed initially in Escherichia coli and shown to produce a 28 kD, phytochrome-binding binding scFV protein. Transgenic tobacco plants expressing the scFV gene were also found to produce a functional scFV protein, and seeds from transgenic R1 progeny displayed aberrant phytochrome-dependent germination. The scFV from transgenic tobacco could be isolated, to near homogeneity, by a single phytochrome-Sepharose affinity chromatography step.

Undergraduate teaching of hypnotherapy and its uses in the United States.

American dental students are able to study hypnotherapy. In Britain no such facility exists. In order to ascertain the feasibility of introducing hypnotherapy into the undergraduate curriculum in Britain I attended an elective in clinical hypnosis at the University of California, Los Angeles.