Size of bacterial ice-nucleation sites measured in situ by radiation inactivation analysis

Four bacterial species are known to catalyze ice formation at temperatures just below 0 degrees C. To better understand the relationship between the molecular structure of bacterial ice-nucleation site(s) and the quantitative and qualitative features of the ice-nucleation-active phenotype, we determined by gamma-radiation analysis the in situ size of ice-nucleation sites in strains of Pseudomonas syringae and Erwinia herbicola and in Escherichia coli HB101 carrying the plasmid pICE1.1 (containing a 4-kilobase DNA insert from P. syringae that confers ice-nucleation activity). Lyophilized cells of each bacterial strain were irradiated with a flux of gamma radiation from 0 to 10.2 Mrad (1 Mrad = 10(6) J/kg). Differential concentrations of active ice nuclei decreased as a first-order function of radiation dose in all strains as temperature was decreased from -2 degrees C to -14 degrees C in 1 degrees C intervals. Sizes of ice nuclei were calculated from the gamma-radiation flux at which 37% of initial ice nuclei active within each 1 degrees C temperature interval remained. The minimum mass of a functional ice nucleus, active only between -12 degrees C and -13 degrees C, was about 150 kDa for all strains. The size of ice nuclei increased logarithmically with increasing temperature from -12 degrees C to -2 degrees C, where the estimated nucleant mass was 19,000 kDa. The ice nucleant in these three bacterial species may represent an oligomeric structure, composed at least in part of an ice gene product that can self-associate to assume many possible sizes.