Department of Genetics, Faculty of Science, Kasetsart University, Chatuchak, Bangkok, Thailand.
Lett Appl Microbiol. 2010 May;50(5):530-6. doi: 10.1111/j.1472-765X.2010.02835.x. Epub 2010 Mar 17.
To develop an intergeneric conjugation system for rimocidin-producing Streptomyces rimosus.
High efficiencies of conjugation [10(-2)-10(-3) transconjugants/recipient colony forming units (CFU)] were obtained when spores of S. rimosus were heat treated at 40 degrees C for 10 min prior to mixing with E. coli ET12567(pUZ8002/pIJ8600) as donor. Mycelium from liquid grown cultures of S. rimosus could also be used as recipient instead of spores, with 24-h cultures giving optimal results. TSA (Oxoid) medium containing 10 m mol l(-1) MgCl(2) was the preferred medium for conjugation. Southern hybridization was used to confirm that transconjugants of S. rimosus contained a single copy of pIJ8600 integrated at a unique chromosomal attachment site (attB). The transconjugants exhibited a high stability of plasmid integration and showed strong expression of green fluorescent protein when using pIJ8655 as the conjugative vector.
Intergeneric conjugation between E. coli and S. rimosus was achieved at high efficiency using both spores and mycelium.
The conjugation system developed provides a convenient gene expression system for S. rimosus R7 and will enable the genetic manipulation of the rimocidin gene cluster.
开发一种用于产生利莫西丁的里莫斯链霉菌的种间接合系统。
在将里莫斯链霉菌孢子与作为供体的大肠杆菌 ET12567(pUZ8002/pIJ8600)混合之前，将孢子在 40°C 下热处理 10 分钟，可以获得高效率的接合[10(-2)-10(-3)转导子/受体集落形成单位 (CFU)]。也可以使用来自里莫斯链霉菌液体生长培养物的菌丝体代替孢子作为受体，24 小时培养物给出最佳结果。含有 10 m mol l(-1)MgCl(2)的 TSA (Oxoid)培养基是用于接合的首选培养基。Southern 杂交用于确认里莫斯链霉菌的转导子含有单个整合在独特的染色体附着位点 (attB)处的 pIJ8600 拷贝。转导子表现出质粒整合的高稳定性，并且当使用 pIJ8655 作为共轭载体时，强烈表达绿色荧光蛋白。
使用孢子和菌丝体，在大肠杆菌和里莫斯链霉菌之间实现了高效的种间接合。
开发的接合系统为 R7 里莫斯链霉菌提供了一种方便的基因表达系统，并将使 rimocidin 基因簇的遗传操作成为可能。
