RESUMEN
Actinoplanes teichomyceticus produces teicoplanin, which is a glycopeptide antibiotic for Gram-positive pathogenic bacteria and methicillin-resistant Staphylococcus aureus (MRSA). For a molecular genetic study of A. teichomyceticus, an effective transformation method using the conjugal transfer of DNA from E. coli to spores of A. teichomyceticus was established for the first time, based on the bacteriophage varphiC31 att/int system, in the genus of Actinoplanes. The high frequency of transconjugation was obtained on MS medium containing 40 mM MgCl(2), using 1.25 x 10(8) E. coli donor cells and 10(5) spores without a heat treatment. In addition, by cloning and sequencing the attB site A. teichomyceticus was shown to contain a single attB site within an ORF coding for a pirin homolog. Also, its attB site sequence showed high homology to that of Streptomyces lividans, unlike the case of Kitasatospora setae despite being a non-Streptomyces actinomycete, which seems to be closely related to the high transconjugation frequency of A. teichomyceticus.
Asunto(s)
Sitios de Ligazón Microbiológica/genética , Fagos de Bacillus/genética , Conjugación Genética , ADN Bacteriano/genética , Micromonosporaceae/genética , Teicoplanina , Antibacterianos/metabolismo , Calor , Micromonosporaceae/metabolismo , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , Teicoplanina/biosíntesisRESUMEN
To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage phiC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl(2) without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the phiC31 att/int system.
Asunto(s)
Sitios de Ligazón Microbiológica/genética , Bacteriófagos/genética , Streptomyces/genética , Bacteriófagos/enzimología , Secuencia de Bases , Southern Blotting , Conjugación Genética , Vectores Genéticos/genética , Integrasas/genética , Integrasas/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Homología de Secuencia de Aminoácido , Espiramicina/metabolismo , Streptomyces/metabolismo , Streptomyces/virologíaRESUMEN
A gene encoding a gamma-butyrolactone autoregulator receptor was cloned in to E. coli from Streptomyces ambofaciens producing spiramycin, a macrolide antibiotic used in both veterinary medicine and human medicine. A 714-bp intact receptor gene (saaR) was obtained by PCR and genomic Southern hybridization with the 100-bp PCR product as a probe. To clarify the in vivo function of saaR, a saaR-disrupted strain was constructed by means of homologous recombination, and phenotypes were compared with those of the wild-type strain. The number of saaR-disruptant spores was 4-fold less than that of the wild-type strain. In addition, saaR deletion from the S. ambofaciens chromosome resulted in complete loss of spiramycin production suggesting that saaR is a rare positive regulator, controlling both spiramycin biosynthesis and sporulation.