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 groES) was originally identified as one of the host factors required for phage lambda growth. The classical grpE280 mutation was the only grpE mutation that resulted from the initial screen and shown to specifically block the initiation of lambda DNA replication. Here we report the isolation of several new grpE missense mutations, again using phage lambda 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. Members of the second group (II), including grpE25, grpE66, grpE103, grpE13a, grpE57b, and grpE61, are sensitive to lambda growth at 30 degrees C but resistant at 42 degrees C. All mutations are recessive, since an E. coli grpE null mutant strain carrying these mutant alleles on low-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 changes were identified by sequencing analyses and shown to be dispersed throughout the latter 75% of the grpE coding region. Most of the amino acid changes occur at conserved residues, as judged by sequence comparisons 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.
Department of Cellular, Viral and Molecular Biology, University of Utah School of Medicine, Salt Lake City 84132.
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B
Wu
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D
Ang
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Snavely
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Georgopoulos
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D010582:Bacteriophage lambda / Q000254:growth & development; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000821:virology; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D006360:Heat-Shock Proteins