Subcellular localization and processing of the lytic transglycosylase of the conjugative plasmid R1

Protein P19 encoded by the conjugative resistance plasmid R1, is essential for efficient conjugative DNA transfer and infection by the pilus-specific RNA phage R17, Based on sequence homologies P19 belongs to a family of lyso zyme-like virulence factors which are found in type III and type IV secreti on systems. In this report we describe the processing and subcellular local ization of P19. Pulse-chase experiments were used to demonstrate the proces sing of P19 by the signal peptidase I of Escherichia coli, Translocation of P19 across the inner membrane was shown by gene 19-phoA fusions, Cell frac tionation studies of P19 expressing cells showed the presence of P19 in the membrane compartment, P19 was solubilized with the detergent Sarkosyl indi cating an inner membrane localization. Using sucrose density gradient centr ifugation to separate inner and outer membranes, P19 was found in both memb rane fractions. Taken together, our data suggest that mature P19 is a perip lasmic protein which may be attached to the proposed membrane-spanning DNA transport complex. (C) 2000 Federation of European Biochemical Societies.