Attempts were made to maximize the expression of ice nuclei in Pseudomonas syringae Ti isolated from a tomato leaf. Nutritional starvation for nitrogen, phosphorous, sulfur, or iron but not carbon at 320C, coupled to a shift to 14 to 18'C, led to the rapid induction of type 1 ice nuclei (i.e., ice nuclei active at temperatures warmer than -50C nuclei and the number of bacterial cells in a culture (i.e., ice nucleation frequency) varied with incubation temperature, growth medium composition, culture age, and genotype. Hirano et al. (9) showed that culturing of different Pseudomonas syringae strains under identical conditions results in ice nucleation frequencies at a given temperature, such as -50C, that vary from one in 100 cells to as few as one in 106 cells. The basis for this variability is unknown. Enhanced expression of ice nuclei, especially type 1 nuclei, has been noted for P. syringae cultures grown at 20 to 240C on nutrient agar containing glycerol (22); optimal conditions increased -50C ice nuclei from 1 in 410,000 to 1 in 72 cells. Using a chemically defined medium, Pooley and Brown (31) similarly noted enhanced ice nucleation frequencies and elevated threshold ice nucleation temperatures for cultures of P. syringae grown on solid versus liquid media. For submerged cultures of P. syringae, Pooley and Brown found that the highest threshold ice nucleation temperatures and frequencies were achieved in mid-to late-log-phase cultures grown in a chemically defined medium rather than a complex growth medium. They suggested that components within the complex medium may have inhibitory effects on the expression of ice nuclei, which is maximal late in the growth cycle. Programmed fermentations designed to achieve maximal cell densities and ice nucleation frequencies have taken advantage of this correlation between the temporal expression of ice nuclei in the cell cycle and nutritional growth requirements (18). In this work, maximal ice nucleation frequencies at -50C, approaching one active nucleus in 10 cells to one in every cell in the population, were achieved for submerged cultures of P. syringae. This result was accomplished by supplying stoichiometrically limiting quantities of nitrogen 4062 on May 9, 2018 by guest

The stability of the ice nucleation activity (INA) and viability of INA Pseudomonas syringae 31a, used as an ice nucleator in the manufacture of synthetic' snow, was determined in snow. The viability of P. syringae 1-2b, a rifampin-resistant mutant selected from strain 31a to improve recovery from test samples, was determined in laboratory tests of three alpine soil and water sampl'es from three different sources.-Snow samples were exposed to environmental conditions or held in'darkness at-20°C. Samples of soil and water were maintained in darkness at 0, 7.5, or 15°C. Parent strain 31a INA decreased significantly (>99.0%) in snow exposed to sunlight and freeze-thaw, while the INA of the cell population in snow held in darkness at-20°C remained essentially unchanged. No viable strain 31a was detected in snow exposed to the environment after 7 days, while the viability of strain 31a in snow held in darkness at-20°C decreased to <3% of the original inoculation at the test conclusion. Mutant strain 1-2b viability was undetectable or had decreased significantly 19 days postinoculation in soil samples held at 0 or 15°C. In contrast, 1-2b viability remained'detectable at low levels for the duration of the test in soils held at 7.5°C. The 1-2b population demonstrated a significantly longer half-life in peatlike soil than in the loam soils tested. The rate of decrease in 1-2b' viability was essentially the same in the three alpine water samples tested with respect to water temperature and sample location.