RESUMEN
BACKGROUND: The granaticins are members of the benzoisochromanequinone class of aromatic polyketides, the best known member of which is actinorhodin made by Streptomyces coelicolor A3(2). Genetic analysis of this class of compounds has played a major role in the development of hypotheses about the way in which aromatic polyketide synthases (PKSs) control product structure. Although the granaticin nascent polyketide is identical to that of actinorhodin, post-PKS steps involve different pyran-ring stereochemistry and glycosylation. Comparison of the complete gene clusters for the two metabolites is therefore of great interest. RESULTS: The entire granaticin gene cluster (the gra cluster) from Streptomyces violaceoruber T-22 was cloned on either of two overlapping cosmids and expressed in the heterologous host, Streptomyces coelicolor A3(2), strain CH999. Chemical analysis of the recombinant strains demonstrated production of granaticin, granaticin B, dihydrogranaticin and dihydrogranaticin B, which are the four known metabolites of S. violaceoruber. Analysis of the complete 39,250 base pair sequence of the insert of one of the cosmids, pOJ466-22-24, revealed 37 complete open reading frames (ORFs), 15 of which resemble ORFs from the act (actinorhodin) gene cluster of S. coelicolor A3(2). Among the rest, nine resemble ORFs potentially involved in deoxysugar metabolism from Streptomyces spp. and other bacteria, and six resemble regulatory ORFs. CONCLUSIONS: On the basis of these resemblances, putative functional assignments of the products of most of the newly discovered ORFs were made, including those of genes involved in the PKS and tailoring steps in the biosynthesis of the granaticin aglycone, steps in the deoxy sugar pathway, and putative regulatory and export functions.
Asunto(s)
Familia de Multigenes/genética , Streptomyces/genética , Streptomyces/metabolismo , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Cósmidos , ADN Bacteriano/biosíntesis , ADN Bacteriano/genética , Glicosilación , Datos de Secuencia Molecular , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/genética , Naftoquinonas/aislamiento & purificación , Naftoquinonas/metabolismo , Sistemas de Lectura Abierta , Plásmidos , ARN de Transferencia/biosíntesis , ARN de Transferencia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
A gene (bar) was identified adjacent to the hrdD sigma factor gene in Streptomyces coelicolor A3(2). The predicted bar product showed 32.2% and 30.4% identity to those of the pat and bar genes of the bialaphos (Bp) producers Streptomyces viridochromogenes and Streptomyces hygroscopicus, respectively; these genes encode phosphinothricin (PPT) N-acetyltransferases that function as enzymes in the Bp biosynthetic pathway and as resistance determinants. The S. coelicolor bar gene conferred high-level resistance to Bp when cloned in S. coelicolor on a high-copy-number vector. Enzymic assay showed that the S. coelicolor bar gene product inactivates PPT by transfer of acetyl groups from acetyl CoA. The S. coelicolor bar gene appears to be expressed from two promoters (p1 and p2) and is divergently transcribed with respect to hrdD. The downstream (barp2) transcript overlaps the hrdDp1 transcript and the upstream (barp1) transcript overlaps both the hrdDp1 and hrdDp2 transcripts. Inactivation of hrdD did not prevent transcription from either bar promoter, indicating that sigma hrdD is not essential for recognition of these sequences.
Asunto(s)
Acetiltransferasas/genética , Antibacterianos/farmacología , Farmacorresistencia Microbiana/genética , Genes Bacterianos , Compuestos Organofosforados/farmacología , Regiones Promotoras Genéticas , Streptomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Genotipo , Datos de Secuencia Molecular , Fenotipo , Plásmidos , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Factor sigma/genética , Streptomyces/efectos de los fármacos , Transcripción GenéticaRESUMEN
The phage growth limitation (Pgl) system of Streptomyces coelicolor confers resistance to phi C31 and its homoimmune phages. The positions of the pgl genes within a 16-kb clone of S. coelicolor DNA were defined by subcloning, insertional inactivation, and deletion mapping. Nucleotide sequencing and functional analysis identified two genes, pglY and pglZ, required for the Pgl+ (phage-resistant) phenotype. pglY and pglZ, which may be translationally coupled, are predicted to encode proteins with M(r)S of 141,000 and 104,000, respectively. Neither protein shows significant similarity to other known proteins, but PglY has a putative ATP/GTP binding motif. The pglY and pglZ genes are cotranscribed from a single promoter which appears to be constitutive and is not induced by phage infection.
Asunto(s)
Proteínas Bacterianas/genética , Bacteriófagos/patogenicidad , Proteínas Portadoras , Genes Bacterianos/genética , Streptomyces/genética , Streptomyces/virología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Mutagénesis , Sistemas de Lectura Abierta/genética , Operón/genética , Fenotipo , Plásmidos/genética , Biosíntesis de Proteínas , Recombinación Genética , Mapeo Restrictivo , Eliminación de Secuencia , Transcripción GenéticaRESUMEN
The multifunctional 6-methylsalicylic acid synthase gene from Penicillium patulum was engineered for regulated expression in Streptomyces coelicolor. Production of significant amounts of 6-methylsalicylic acid by the recombinant strain was proven by nuclear magnetic resonance spectroscopy. These results suggest that it is possible to harness the molecular diversity of eukaryotic polyketide pathways by heterologous expression of biosynthetic genes in an easily manipulated model bacterial host in which prokaryotic aromatic and modular polyketide synthase genes are already expressed and recombined.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Complejos Multienzimáticos/genética , Streptomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos/genética , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multienzimáticos/biosíntesis , Penicillium/enzimología , Penicillium/genética , Plásmidos/genética , Salicilatos/metabolismo , Streptomyces/enzimologíaRESUMEN
A mutation in actVI-ORF1, which controls C-3 reduction in actinorhodin biosynthesis by Streptomyces coelicolor, was complemented by gra-ORF5 and -ORF6 from the granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22. It is hypothesized that, while gra-ORF5 alone is a ketoreductase for C-9, gra-ORF6 gives the enzyme regiospecificity also for C-3.