Identification of important amino acid residues that modulate binding of Escherichia coli GroEL to its various cochaperones

Genetic experiments have shown that the GroEL/GroES chaperone machine of Es cherichia coli is absolutely essential, not only for bacterial growth but a lso for the propagation of many bacteriophages including lambda. The virule nt bacteriophages T4 and RB49 are independent of the host GroES function, b ecause they encode their own cochaperone proteins, Gp31 and CocO, respectiv ely. E. coli groEL44 mutant bacteria do not form colonies above 42 degrees nor do they propagate bacteriophages lambda, T4, or RB49. We found that the vast majority (40/46) of spontaneous groEL44 temperature-resistant colonie s at 43 degrees were due to the presence of an intragenic suppressor mutati on. These suppressors define 21 different amino acid substitutions in GroEL , each affecting one of 13 different amino acid residues. All of these amin o acid residues are located at or near the hinge, which regulates the large en bloc movements of the GroEL apical domain. All of these intragenic supp ressors support bacteriophages lambda, T4, and RB49 growth to various exten ts in the presence of the groEL44 allele. Since it is known that the GroEL4 4 mutant protein does not interact effectively with Gp31, the suppressor mu tations should enhance cochaperone binding. Analogous intragenic suppressor studies were conducted with the groEL673 temperature-sensitive allele.