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Influence of Protein and Ribonucleic Acid Synthesis on the Replication of the Bacteriocinogenic Factor Clo DF13 in Escherichia coli Cells and Minicells

Department of Molecular Genetics, Biological Laboratory, Free University, Amsterdam-Buitenveldert, The Netherlands

ABSTRACT

The influence of ribonucleic acid (RNA) and protein synthesis on the replication of the cloacinogenic factor Clo DF13 was studied in Escherichia coli cells and minicells. In chromosomeless minicells harboring the Clo DF13 factor, Clo DF13 deoxyribonucleic acid (DNA) synthesis is slightly stimulated after inhibition of protein synthesis by chloramphenicol or puromycin and continues for more than 8 h. When minicells were treated with rifampin, a specific inhibitor of DNA-dependent RNA polymerase, Clo DF13 RNA and DNA synthesis appeared to stop abruptly. In cells, the Clo DF13 factor continues to replicate during treatment with chloramphenicol long after chromosomal DNA synthesis ceases. When rifampin was included during chloramphenicol treatment of cells, synthesis of Clo DF13 plasmid DNA was blocked completely. Isolated, supercoiled Clo DF13 DNA, synthesized in cells or minicells in the presence of chloramphenicol, appeared to be sensitive to ribonuclease and alkali treatment. These treatments convert a relatively large portion of the covalently closed Clo DF13 DNA to the open circular form, whereas supercoiled Clo DF13 DNA, isolated from non-chloramphenicol-treated cells or minicells, is not significantly affected by these treatments. These results indicate that RNA synthesis and specifically Clo DF13 RNA synthesis are involved in Clo DF13 DNA replication and that the covalently closed Clo DF13 DNA, synthesized in the presence of chloramphenicol, contains one or more RNA sequences. De novo synthesis of chromosomal and Clo DF13-specific proteins is not required for the replication of the Clo DF13 factor. Supercoiled Clo DF13 DNA, isolated from a polA107 (Clo DF13) strain which lacks the 5' 3' exonucleolytic activity of DNA polymerase I, is insensitive to ribonuclease or alkali treatment, indicating that in this mutant the RNA sequences are still removed from the RNA-DNA hybrid.