Altered DNA-protein relaxation complex in a replication mutant of plasmid ColE1

Approximately 200,000 clones of Escherichia coli carrying mutagen-treated colicinogenic plasmid E1 (ColE1) were examined for irreversible loss of the plasmid at 43 degrees. Thirty of these clones that appeared to be most defective in plasmid DNA replication at the non-permissive temperature were selected for the study of: (a) the kinetics of plasmid and chromosomal DNA replication during a temperature shift in either the presence or absence of chloramphenicol; (b) the temperature stability of the plasmid DNA-protein relaxation complex; and (c) the temperature effect on F-promoted conjugal transfer. Two mutant plasmids, pJC307 and pJC301, showed defects in their relaxation complex. The relaxation complex of pJC307 exhibited an altered temperature stability in vitro. Reversion to temperature resistant replication resulted in four out of five cases in a concomitant change in the temperature stability of the relaxation complex. Conjugal mobility of this mutant was not markedly reduced at the permissive or non-permissive temperature. Plasmid pJC301 could not be isolated in the form of a relaxation complex and it was very poorly mobilized in an F'-promoted conjugation. These results indicate that the ColE1 plasmid codes for at least one of the proteins of the relaxation complex and that the relaxation complex is involved in ColE1 DNA replication. In addition, the properties of the mutant plasmid pJC301 are consistent with a role for the complex in the mobilization of ColE1 during conjugation.