ISOLATION AND CHARACTERIZATION OF POINT MUTATIONS IN THE ESCHERICHIA-COLI GRPE HEAT-SHOCK GENE

The Escherichia coli grpE gene (along with dnaK, dnaJ, groEL, and groE S) was originally identified as one of the host factors required for p hage lambda growth. The classical grpE280 mutation was the only grpE m utation that resulted from the initial screen and shown to specificall y block the initiation of lambda DNA replication. Here we report the i solation of several new grpE missense mutations, again using phage lam bda resistance as a selection. All mutants fall into two groups based on their temperature-dependent phenotype for lambda growth. Members of the first group (I), including grpE17 and grpE280, which was obtained again, are resistant to lambda growth at both 30 and 42 degrees C. Me mbers of the second group (II), including grpE25, grpE66, grpE103, grp E13a, grpE57b, and grpE61, are sensitive to lambda growth at 30 degree s C but resistant at 42 degrees C. All mutations are recessive, since an E. coli grpE null mutant strain carrying these mutant alleles on lo w-copy-number plasmids are sensitive to infection by the lambda grpE() transducing phage. Both group I and group II mutants are temperature sensitive for E. coli growth above 42 degrees C. The nucleotide chang es were identified by sequencing analyses and shown to be dispersed th roughout the latter 75% of the grpE coding region. Most of the amino a cid changes occur at conserved residues, as judged by sequence compari sons between E. coli and other bacterial and yeast GrpE homologs. The isolation of these new mutations is the first step toward a structure- function analysis of the GrpE protein.