Localization of plasmid DNA in living Streptomyces lividans mycelium

Abstract

The Gram-positive soil bacterium Streptomyces grows by apical tip extension, forming a multiply branched mycelium. This mode of growth poses a problem for the proper distribution of the genetic material, i.e. the chromosome and plasmids. So far, the position of a plasmid has not been determined in a mycelial organism. To elucidate the localization of the single copy number plasmid SCP2 in living S. lividans mycelium in comparison with that of the chromosome, a fluorescent repressor operator system (FROS) was applied. A tetO array consisting of 120 binding sites was inserted into a bifunctional SCP2 derivative (SCP2tetO) or integrated into the central part (nt. 5327700) of the S. lividans chromosome. Integration of a gene encoding a TetR-mCherry fusion protein resulted in bright fluorescence foci, indicating the positions of individual copies of the plasmid or the chromosomal region, marked with the tetO array. Still-images of hyphae and time-lapse imaging of SCP2tetO and the chromosome revealed a very similar localization pattern, with the tip-proximal copy always following the tip at a constant distance of 2.21µm; ± 0.99µm (SCP2tetO) and 3.69µm; ± 2.86µm (chromosome). The localization pattern of SCP2tetO depended on the ParABSCP2 partitioning system. Deletion of parASCP2 reduced the number of visible plasmid spots. These spots had a higher fluorescence intensity compared to those of the parent plasmid SCP2tetO, suggesting that, following replication, the plasmid copies could not separate in the absence of ParASCP2. In addition, ParABSCP2 seemed to be required to anchor SCP2tetO to branching sites and hyphal tips, since SCP2tetOΔparA was detected only very rarely at branching sites and tips. The FROS system also allowed observation of conjugative SCP2tetO transfer and the mobilization of chromosomal DNA to a recipient strain, encoding eGFP and the TetRmcherry fusion protein. These studies suggested that the angle of the hyphal contact site was important for conjugative transfer. Plasmid transfer was only observed if donor and recipient hyphae hit each other at an angle above 59 °, while no transfer was observed when the angle was below 27.5 °. Using FROS the conjugative transfer of chromosomal DNA and its establishment and subsequent spreading in the recipient mycelium was visualized. This is the first report demonstrating the conjugative transfer of chromosomal DNA by fluorescence imaging.