ISOLATION AND CHARACTERIZATION OF HYDROXYLAMINE-INDUCED MUTATIONS IN THE ERWINIA-HERBICOLA ICE NUCLEATION GENE THAT SELECTIVELY REDUCE WARMTEMPERATURE ICE NUCLEATION ACTIVITY

Cells of ice nucleation active bacterial species catalyse ice formatio n over the temperature range of -2 to -12-degrees-C. Current models of ice nucleus structure associate the size of ice nucleation protein ag gregates with the temperature at which they catalyse ice formation. To better define the structural features of ice nucleation proteins resp onsible for the functional heterogeneity of ice nuclei within a geneti cally homogeneous collection of cells we used in vitro chemical mutage nesis to isolate mutants with reduced ability to nucleate ice at warm assay temperatures but which retain normal or near normal nucleation a ctivity at cold temperatures (WIND, i.e. warm ice nucleus-deficient mu tants). Nearly haff of the mutants obtained after hydroxylamine mutage nesis of the iceE gene from Erwinia herbicola had this phenotype. The phenotypes and location of lesions on the genetic map of iceE were det ermined for a number of mutants. All WIND mutations were restricted to the portion of iceE encoding the repetitive region of the polypeptide . DNA sequencing of two WIND mutants revealed single nucleotide substi tutions changing a conserved serine or glycine residue to phenylalanin e and serine, respectively. The implications of these findings in stru cture/function models for the ice nucleation protein are discussed.