Functional and mutational analysis of p19, a DNA transfer protein with muramidase activity.
Protein P19 encoded by the conjugative resistance plasmid R1 has been identified as being one member of a large family of muramidases encoded by bacteriophages and by type III and type IV secretion systems. We carried out a mutational analysis to investigate the function of protein P19 and used in vivo complementation assays to test those of several P19 mutants. The results indicated that conserved residues present in the presumed catalytic center of P19 are absolutely essential for its function in conjugation of plasmid R1 and infection by the RNA phage R17. Overexpression of protein P19 in an 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 culture 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 microscopy. Typically, P19-overexpressing cells showed bulges protruding from the cell surfaces. Our interpretation is that these protrusions arose from a localized and spatially confined disruption of the bacterial cell wall. To our knowledge such an effect has not previously been documented for any member of the lytic transglycosylase family. From the data presented here, we conclude that protein P19 possesses the proposed localized peptidoglycan-hydrolyzing activity. This activity would be a prerequisite for efficient penetration of the cell envelope by the DNA translocation complex encoded by the conjugative plasmid.
Bayer
M
M
Institut für Molekularbiologie, Biochemie und Mikrobiologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
Iberer
R
R
Bischof
K
K
Rassi
E
E
Stabentheiner
E
E
Zellnig
G
G
Koraimann
G
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D003227:Conjugation, Genetic; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000821:virology; D005816:Genetic Complementation Test; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011815:R Factors / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D009113:Muramidase