RESUMO
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.
Assuntos
Metionina/genética , Metionina/metabolismo , Streptomyces/genética , Tilosina/biossíntese , Sequência de Aminoácidos , Sequência Conservada , DNA Bacteriano/química , Resistência Microbiana a Medicamentos/genética , Fermentação , Genes Bacterianos , Dados de Sequência Molecular , Família Multigênica , Mapeamento por Restrição , Análise de Sequência de DNA , Streptomyces/metabolismoRESUMO
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.
Assuntos
Antibacterianos/biossíntese , Regulação Fúngica da Expressão Gênica/genética , Streptomyces/genética , Tilosina/biossíntese , Sequência de Aminoácidos , Códon , Análise Mutacional de DNA , DNA Fúngico/biossíntese , DNA Fúngico/genética , Fermentação/genética , Fermentação/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Família Multigênica/genética , Fases de Leitura Aberta , Receptores de GABA-A/biossíntese , Receptores de GABA-A/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Streptomyces/metabolismoRESUMO
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.
Assuntos
Antibacterianos/biossíntese , Streptomyces/genética , Tilosina/biossíntese , Antibacterianos/química , Mapeamento Cromossômico , Cromossomos Bacterianos , DNA Bacteriano , Genes Bacterianos , Teste de Complementação Genética , Glucosamina/análogos & derivados , Glucosamina/biossíntese , Dados de Sequência Molecular , Estrutura Molecular , Família Multigênica , Fases de Leitura Aberta , Homologia de Sequência de Aminoácidos , Streptomyces/enzimologia , Tilosina/químicaRESUMO
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.
Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Metiltransferases/genética , Streptomyces/genética , Tilosina/biossíntese , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/classificação , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Resistência Microbiana a Medicamentos , Genoma Bacteriano , Lincosamidas , Macrolídeos/farmacologia , Metiltransferases/classificação , Dados de Sequência Molecular , Filogenia , Mapeamento por Restrição , Streptomyces/metabolismo , Virginiamicina/farmacologiaAssuntos
Anticorpos Monoclonais/biossíntese , Imunoglobulinas/biossíntese , Plantas Geneticamente Modificadas , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Agricultura , Animais , Sequência de Bases , Humanos , Fragmentos de Imunoglobulinas/biossíntese , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/isolamento & purificação , Transcrição GênicaRESUMO
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.