Fluorescence microscopy of Streptomyces conjugation suggests DNA-transfer at the lateral walls and reveals the spreading of the plasmid in the recipient mycelium.

Abstract

Conjugative DNA-transfer in mycelial streptomycetes is a unique process, manifested on agar plates by the formation of circular growth retardation zones called pocks. Because pock size correlates with the extent of the transconjugant zone, it was suggested that pocks reflect the spreading of the transferred plasmid in the recipient mycelium. However, this concept has not been experimentally proven yet. The use of an eGFP-encoding derivative of the conjugative pIJ303 plasmid and Streptomyces lividans T7-mCherry as recipient enabled us to differentiate donor, recipient and transconjugant hyphae in mating experiments by fluorescence microscopy. Microscopic observation of the conjugation process suggested DNA-transfer via the lateral walls. At the contact sites mCherry was never observed in the donor, indicating that the conjugative DNA-transfer does not involve interfusion of cytoplasms of donor and recipient. The spreading of the transferred plasmid to the older parts of the recipient mycelium was demonstrated. This spreading was impaired when plasmid-encoded spd genes were inactivated. Deletion of the FtsK-like DNA-translocase encoding tra gene from the plasmid and mating experiments with strains containing chromosomal copies of tra either in the donor and/or in the recipient revealed that Tra had an essential role in intramycelial plasmid spreading.