A Carboxy-Terminal Deletion Impairs the Assembly of GroEL and Confers a Pleiotropic Phenotype in Escherichia coli K-12

A series of COOH-terminal deletions of the chaperonin GroEL have been examined for effects in vivo at haploid copy number on the essential requirement of GroEL for cell growth. Strains with a deletion of up to 27 COOH-terminal amino acids were viable, but no viable strain could be isolated with a deletion of 28 or more codons. When substitutions were placed in the COOH-terminal amino acid Val-521 of the 27-amino-acid-deleted (A27) mutant, we found variable effects-Trp and Glu led to inviability, whereas Arg and Gly were viable but slow growing. The effects of the Arg substitution plus deletion (V521RA) were examined in more detail. Whereas the A27 mutant with the wild-type residue Val-521 grew as well as a strain with wild-type GroEL, the V521RA mutant strain (groEL202) exhibited a broad range of phenotypic defects. These include slow growth; filamentous morphology; a defect in plating X; absence of activity of expressed human ornithine transcarbamylase, as seen in other GroEL mutants; and several newly observed defects, such as absence of motility, sensitivity to UV light and mitomycin, a defect in one mode of specialized transduction, and inability to grow on rhamnose. Sucrose gradient analysis of extracts from the V521RA cells showed a substantially reduced level of GroEL sedimenting at the normal 20S position of the assembled tetradecamer and a relatively large amount of more lightly sedimenting subunits. This indicates that the substitution-deletion mutation interferes with oligomeric assembly of GroEL into its functional form. This is discussed in light of the recentl