Interactions between Phage λ Replication Proteins, λ DNA and Minicell Membrane

Abstract

Gentle methods for [Escherichia coli] minicell lysis and lysate fractionation were elaborated: lysis by T4 lysozyme without detergents and fractionation by equilibrium sedimentation in a metrizamide density gradient, both at low ionic strength. In the lysates of phage-.lambda.-infected minicells the .lambda. DNA, trapped at a prereplicative step, appeared in 2 peaks of different buoyant densities: as a membrane-bound and a free .lambda. DNA. The covalently-closed-circular form of .lambda. DNA appeared exclusively in the membrane fraction. The .lambda.-coded proteins, synthesized in .lambda.-infected minicells, appeared in 2 major fractions: as membrane-bound and as free proteins, and in one minor fraction, bound with free .lambda. DNA. Neither .lambda. protein engaged in the initiation of DNA replication was present in the fraction of free proteins: the P-gene product was membrane-associated and the O-gene product formed a complex with free .lambda. DNA. The effect of high ionic strength (KCl) and of detergents (Triton X-100 and sarcosyl) on the binding of replication proteins with .lambda. DNA and with the membrane was studied. The non-ionic detergent, Triton X-100 caused displacement of a part of .lambda. DNA from the membrane to the free .lambda. DNA peak; both .lambda. replication proteins were bound with free .lambda. DNA. The binding of the O protein with .lambda. DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.