Functional and mutational analysis of P19, a DNA transfer protein with muramidase activity

Protein P19 encoded by the conjugative resistance plasmid RI has been ident ified as being one member of a large family of muramidases encoded by bacte riophages and by type III and type IV secretion systems. We carried out a m utational analysis to investigate the function of protein P19 and used in v ivo complementation assays to test those of several P19 mutants. The result s indicated that conserved residues present in the presumed catalytic cente r of P19 are absolutely essential for its function in conjugation of plasmi d RI and infection by the RNA phage R17. Overexpression of protein P19 in a n early growth phase resulted in a massive lysis of Escherichia coli cells in liquid culture, as indicated by a rapid and distinct decrease in cell cu lture densities after induction, Change of the proposed catalytic glutamate at position 44 to glutamine completely abolished this effect. P19-induced cell lysis was directly shown by transmission and scanning electron microsc opy. Typically, P19-overexpressing cells showed bulges protruding from the cell surfaces. Our interpretation is that these protrusions arose from a lo calized and spatially confined disruption of the bacterial cell wall. To ou r knowledge such an effect has not previously been documented for any membe r of the lytic transglycosylase family. From the data presented here, we co nclude that protein P19 possesses the proposed localized peptidoglycan-hydr olyzing activity. This activity would be a prerequisite for efficient penet ration of the cell envelope by the DNA translocation complex encoded by the conjugative plasmid.