Isolation, Purification and Characterization of Nucleoids from <i>Synechococcus elongatus</i> PCC 7942

Abstract

The genomic DNA of bacteria is highly compacted in one or a few bodies known as nucleoids. In order to understand the overall configuration and physiological activities of the cyanobacterial nucleoid under various growth conditions and the role(s) of each nucleoid protein in clock function, thylakoid membrane-associated nucleoids from the Synechococcus elongatus (se) PCC 7942 strain were isolated and purified in presence of spermidine at low salt concentrations by sucrose density gradient centrifugation. The sedimentation rates, protein/DNA composition and microscopic appearances as well as variation in structural components of clock proteins from the isolated nucleoids were compared under identical conditions. Microscopic appearances of the nucleoids were consistent with the sedimentation profiles. The nucleoid structure in the wild type was more tightly compacted than that in the KaiABC mutant strain. Western immunoblot analyses revealed that the KaiC was associated with the nucleoid fraction whereas maximum KaiA was localized in the cytosolic fraction, supposedly in association with the translation machinery.