Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Kitasatospora setae , a bafilomycin B 1 producer

Abstract

An effective transformation procedure for Kitasatospora setae was established based on transconjugation from Escherichia coli ET12567 (pUZ8002) using a phi C31-derived integration vector, pSET152, containing oriT and attP fragments. While no transconjugation was observed under the standard transconjugation conditions for Streptomyces species, sufficient transconjugation (>1 x 10(-6)) was achieved on ISP4 medium containing 30 mM MgCl(2) using a 25- to 125-fold excess of E. coli donor cells. In addition, the sequence and location of the chromosomal integration site attB of K. setae was identified for the first time in genera of non- Streptomyces actinomycetes. K. setae contains a single phi C31 attB site. Similar to the case of Streptomyces species, the attB site of K. setae is present within an ORF encoding a pirin-homolog, but the K. setae-attB sequence deviates slightly from the consensus sequence of Streptomyces attB sequences.

Characterization of bafilomycin biosynthesis in Kitasatospora setae KM-6054 and comparative analysis of gene clusters in Actinomycetales microorganisms

“…The recombinant DNA may be introduced into K. setae by an E. coli/ Streptomyces conjugation using an IncP-plasmid (RK2) origin transfer. 34,35 However, we found that the transformation of K. setae KM-6054 was improved by electroporation (approximately 10-to 100-fold higher than the conjugation with E. coli and IncP-plasmid). K. setae generated submerged spores in the liquid culture and DNA was introduced into the submerged spores by electroporation.…”

Optimization of transconjugation and characterization of attB integration site for Streptomyces cinnamoneus producing transglutaminase

“…In the case of Kitasatospora setae conjugation, the use of 2.5 × 10 7 E. coli as donor yielded no exconjugants, but a further increase in the number of E. coli facilitated the conjugal transfer of K. setae (Choi et al 2004). As shown in Table 2, when 1 × 10 4 spores were used as the recipient for conjugation, the transconjugation frequency was proportionately improved with increasing numbers of E. coli donor.…”

“…Parameters affecting transconjugation efficiency To select an optimal medium for the conjugal transfer of S. cinnamoneus, four media (AS-1, ISP2, ISP4, and MS) were tested because the effect of medium for the successful conjugal transfer of actinomycetes is absolute (Bierman et al 1992;Flett et al 1997;Choi et al 2004). AS-1, ISP4, and MS are representative media for the conjugal transfer of actinomycetes and ISP2 is an optimal medium for growth and spore formation of S. cinnamoneus.…”

“…ciency of actinomycetes (Kieser et al 2000). However, because the optimal concentration of the incorporated MgCl 2 differs between host strains (Choi et al 2004), its concentration was investigated for conjugation of S. cinnamoneus. When 0-60 mM MgCl 2 was added to MS, the transconjugation frequency was increased by all concentrations of MgCl 2 and proportionately improved until 50 mM.…”

“…Studies using various strains have determined that the mixing ratio of the number of recipient spore and E. coli donor cells profoundly affects the transconjugation efficiency of actinomycetes (Choi et al 2004;Kim et al 2008). In the case of Kitasatospora setae conjugation, the use of 2.5 × 10 7 E. coli as donor yielded no exconjugants, but a further increase in the number of E. coli facilitated the conjugal transfer of K. setae (Choi et al 2004).…”