N-126. Natural Diversity and Experimental Evolution of Environmental Stress Tolerance in Marine Bacteria

A. C. Materna1, S. A. Clarke1, C. Cruz1, X. Gao1, E. J. Alm1,2,3;
1Massachusetts Inst. of Technology, Cambridge, MA, 2The Virtual Inst. of Microbial Stress and Survival, Berkeley, CA, 3The Broad Inst. of MIT and Harvard Univ., Cambridge, MA.

Genome sequencing has revealed extensive genetic variation within bacterial species and among co-existing bacteria. Using marine Vibrio strains as a model system, we investigate to what extent observed sequence diversity corresponds to measurable differences in salinity and temperature tolerance phenotypes, two ecologically important factors for this group of organisms. Using directed evolution, we quantify how malleable these phenotypes are with respect to a small number of mutation events. We have designed two-dimensional gradients in 24 cm square dishes containing solid growth medium to monitor temperature and salinity tolerances over a broad range of both factors. Growth patterns indicate the strain-specific minimum and maximum tolerances and interactions between the factors (salinity and temperature). We compared the specific boundaries of growth for multiple strains of Vibrio splendidus and V. alginolyticus. While the obtained profiles differ in their shape and limits, some consistent features appear. Tolerance to increasing salinities correlated positively with temperature tolerance. However, higher salinity constrained the limits of temperature tolerance, so that the maximum salinity tolerance occurred at intermediate temperatures. Similarly, growth at higher temperatures led to a tradeoff, limiting the range of salinity tolerance. Interestingly, at high salinities, low temperatures tended to suspend growth, leaving viable cells that could be regenerated when the temperature gradient was removed, while higher temperatures led to killing. In addition to comparing related environmental isolates, this method was further applied to study differences between parental and evolved strains. Serial application of 106 cells/cm2 to the solid medium gradients enabled selection for spontaneous, more tolerant mutants. In future work, this integrated ecological and experimental approach will be combined with genome re-sequencing to draw connections between genetic diversity and ecologically relevant phenotypes and tradeoffs.