Agrobacterium-mediated T-DNA transfer and integration into the chromosome of Streptomyces lividans

Summary Agrobacterium tumefaciens is the prototype of a prokaryotic organism transmitting DNA across natural kingdom barriers into higher cells. In nature, a specific segment (T-DNA) of the resident Ti plasmid is transferred from this bacterium into plant cells and integrated into the plant cell genome. Expression of the integrated oncogenes contained in the T-DNA results in the tumour disease known as crown gall. Besides plants, the range of transformable recipients is broad and includes fungi and mammalian cells. We now show further extension of this host range, whereby the actinomycete Streptomyces lividans is also a recipient of the T-DNA. A. tumefaciens cells containing a binary vector system with a vir helper plasmid, pUCD2614, and a T-DNA donor plasmid, pUCD5801, were co-cultured with S. lividans hyphae. A. tumefaciens-S. lividans aggregate when the vir genes are induced with acetosyringone, resulting in the transfer of the T-DNA, as evidenced by the formation of transconjugants containing T-DNA genetic markers and the appearance of the T-DNA in these transconjugants. Close examination of the interacted cells revealed a presumably coiled thread-like interconnection with an average width of approximately 30 nm between A. tumefaciens and S. lividans. This interconnecting structure is dependent on virB genes and appears only under the same conditions as that required for T-pilus formation. Insertion of the T-DNA via A. tumefaciens into the S. lividans genome provides a useful genetic tool for generating novel mutants.