Uptake of cloacin DF13 by susceptible cells: removal of immunity protein and fragmentation of cloacin molecules.
Monoclonal antibodies (MAb) directed against different epitopes on the equimolar complex of cloacin and immunity protein (cloacin DF13) were isolated, characterized, and used to study the uptake of cloacin DF13 by susceptible cells. Four MAbs recognized the amino-terminal part, one MAb recognized the central part, and three MAbs recognized the carboxyl-terminal part of the cloacin molecule. Three MAbs reacted with the immunity protein. Five MAbs inhibited the lethal action of cloacin DF13, but none of the MAbs inhibited the binding of cloacin DF13 to its purified outer membrane receptor protein or the in vitro inactivation of ribosomes. Binding of cloacin DF13 to susceptible cells cultured in broth resulted in a specific, time-dependent dissociation of the complex and a fragmentation of the cloacin molecules. Increasing amounts of immunity protein were detected in the culture medium from about 20 min after the addition of cloacin DF13. Cloacin was fragmented into two carboxyl-terminal fragments with relative molecular masses of 50,000 and 10,000. The larger fragment was detected 5 min after the binding of the bacteriocin complex to the cells. The smaller fragment was detected after 10 min. Both fragments were associated with the cells and could not be detected in the culture supernatant fraction. Cells grown in brain heart infusion were much less susceptible to cloacin DF13 than cells grown in broth, although they possessed a similar number of outer membrane receptor molecules. This decreased susceptibility correlated with a decreased translocation, dissociation, and fragmentation of cloacin DF13.
Krone
W J
WJ
de Vries
P
P
Koningstein
G
G
de Jonge
A J
AJ
de Graaf
F K
FK
Oudega
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000378:metabolism*; D001430:Bacteriocins / Q000378:metabolism*; D002989:Cloacin / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D003470:Culture Media; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D001430:Bacteriocins; D003470:Culture Media; D000939:Epitopes; D029968:Escherichia coli Proteins; C027355:cloacin DF13 protein, E coli; D002989:Cloacin