Michaels K, Mei J, Firshein W
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459.
Plasmid. 1994 Jul;32(1):19-31. doi: 10.1006/plas.1994.1040.
Previous results of experiments in which plasmid-encoded proteins were selectively labeled in ultraviolet sensitive "maxicell" mutants suggested that the essential initiation proteins of RK2 (33 and 43 kDa) were bound to the inner membrane of Escherichia coli (D. Kostyal et al., 1989, Plasmid 21, 226-237). However, in the present studies using a specific polyclonal antibody against the TrfA initiation proteins, significant levels of these proteins were also detected for the first time in the outer membrane fraction as well as the inner membrane fraction. Only in the cytosol fraction were the initiation proteins relatively absent. In order to determine whether initiation and replication were also associated with either or both of the membrane fractions, it was necessary to develop a replicating system more active than the one previously extracted from minicell membranes, which did not separate the membrane into its component parts (J. A. Kornacki and W. Firshein, 1986, J. Bacteriol. 167, 319-326). In addition, it was also necessary to devise an extraction procedure that did not degrade the supercoil DNA template during the separation of the inner from the outer membrane fraction. Both criteria were met, first by the use of maxicells containing a miniplasmid derivative of RK2 and second by disrupting cell envelopes in the French pressure cell using low pressure. Under these conditions, not only were the two major membrane fractions separated successfully from the cytosol fraction, but supercoil DNA template was also preserved in both fractions, detergents were avoided, and replication was significantly higher than that described in the earlier experiments. TrfA-dependent initiation of DNA replication was associated primarily with the inner membrane fraction.
