IN-VITRO REPLICATION OF CYANOBACTERIAL PLASMIDS FROM SYNECHOCYSTIS PCC-6803

Little knowledge of DNA replication in cyanobacteria is available. In this study, we report the development and characterization of an in vi tro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803. This system (fracti on III) was isolated at high salt concentrations and partially purifie d on a heparin-agarose column. DNA polymerases in Synechocystis 6803 a ppeared to be associated with membranes and could be released by the a ddition of ammonium sulfate to 20% saturation. DNA synthesis in fracti on III was dependent on the addition of cyanobacterial plasmids isolat ed from the same strain. The in vitro replication products consist mos tly of the supercoiled form of the plasmids. Unlike replication of man y Escherichia call plasmids, replication of cyanobacterial plasmids di d not require added ATP, was not inhibited by omission of the ribonucl eotides, and was insensitive to the RNA polymerase inhibitor rifampici n and the gyrase inhibitor novobiocin, but was inhibited by ethidium b romide. These data suggest that RNA may not be involved in the initiat ion of replication of cyanobacterial plasmids from Synechocystis 6803. In addition, intermediates of replication have been detected by two-d imensional gel electrophoresis. Density labeling experiments also indi cate that cyanobacterial plasmid synthesis in vitro occurs by a semico nservative replication. (C) 1994 Academic Press, Inc.