Experimental evolution enhances thermal tolerance in an already highly thermostable bacteriophage

Thermal stability influences bacteriophage survival during extracellular phases, when virions must persist outside the protective environment of the host under fluctuating and potentially damaging environmental conditions. Because many random mutations are destabilizing, further gains in stability may in some systems be less accessible than losses of stability. We examined this question using Salmonella Phage SA01, an exceptionally thermostable lytic bacteriophage that shows minimal loss of infectivity after prolonged exposure to 65 °C. We subjected SA01 populations to experimental evolution using ethyl methanesulfonate mutagenesis and progressively increasing thermal selection (73–84 °C). Relative to the ancestral population, the evolved SA01 population showed improved survival at 70 and 72 °C, increased cumulative viability and slower infectivity loss under heat stress. The adaptive phenotype emerged early, with a significant improvement already evident by round 2, and appeared to stabilize after round 5; importantly, an independently repeated evolution experiment reproduced similar phenotypic outcome. Sequencing of a thermostable clone identified a dominant EMS-compatible mutation in the NinH gene, resulting in an Ala25Thr substitution, suggesting a potential association with the enhanced thermal tolerance phenotype. Improved thermal tolerance was not accompanied by detectable changes in host range, bacteriolytic dynamics, efficiency of plating, adsorption kinetics, or growth parameters under the tested laboratory conditions. Experimental evolution successfully enhanced the thermal tolerance of an already highly thermostable bacteriophage without altering key infectivity traits. A NinH mutation was identified in the thermostable clone suggesting a potential association with the observed increase in thermal tolerance.