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The role of the Streptomyces ParB-DNA complex in chromosome segregation and regulation of DNA replication

Final Activity Report Summary - SCPARB-DNA (The role of the Streptomyces ParB-DNA complex in chromosome segregation and regulation of DNA replication)

Bacterial chromosome segregation has recently been shown to be an active process involving number of proteins including ParA (ATPase) and ParB (DNA binding protein) homologues. The proposed project focused on chromosome segregation in the mycelial microorganism Streptomyces coelicolor. Streptomyces spp. are Gram-positive soil bacteria that produce many valuable antibiotics and other secondary metabolites. During mycelial growth of S. coelicolor chromosome segregation occurs only during the formation of chains of unigenomic spores from multigenomic aerial hyphae.

Disruption of the parAB operon in S. coelicolor leads to disturbed chromosome segregation into spores. ParB is DNA binding protein that recognises parS sites clustered in S. coelicolor chromosome around the oriC region. ParB binding results in formation of massive nucleoprotein complex. Formation of such complexes has been visualised in vivo with the application of fluorescence microscopy and in vitro with the electron microscope.

There are some suggestions that formation of such complexes may influence accessibility of DNA for other proteins and particularly provide steric hindrance for replication proteins binding in proximal oriC region.

One of the aims of the project was to test this hypothesis. We have constructed strain that expresses one of DNA polymerase III subunits (b-clamp) fused to green fluorescent protein enabling visualisation of replisomes.

Our results suggest that indeed, replication rate is accelerated in the parB deletion mutant. We have also shown that ParA is an important factor influencing formation of ParB complex. Overproduction of ParA promoted formation of ParB complexes and also resulted in slower growth probably due to DNA replication inhibition. Additionally we have conducted the search for other proteins interacting with the ParB complexes and their organiser ParA using in vivo bacterial two hybrid system and in vitro biochemical assay (fishing experiment). Several potential candidates have been identified and will be subject of further analysis.