Genetic Basis of Evolutionary Adaptation by Escherichia Coli to Stressful Cycles of Freezing, Thawing and Growth
Sleight, Sean C., Orlic, Christian, Schneider, Dominique, Lenski, Richard E., Genetics
Microbial evolution experiments offer a powerful approach for coupling changes in complex phenotypes, including fitness and its components, with specific mutations. Here we investigate mutations substituted in 15 lines of Escherichia coli that evolved for 1000 generations under freeze-thaw-growth (FTG) conditions. To investigate the genetic basis of their improvements, we screened many of the lines for mutations involving insertion sequence (IS) elements and identified two genes where multiple lines had similar mutations. Three lines had IS150 insertions in cls, which encodes cardiolipin synthase, and 8 lines had IS150 insertions in the uspA-uspB intergenic region, encoding two universal stress proteins. Another line had an 11-bp deletion mutation in the cls gene. Strain reconstructions and competitions demonstrated that this deletion is beneficial under the FTG regime in its evolved genetic background. Further experiments showed that this cls mutation helps maintain membrane fluidity after freezing and thawing and improves freeze-thaw (FT) survival. Reconstruction of isogenic strains also showed that the IS150 insertions in uspA/B are beneficial under the FTG regime. The evolved insertions reduce uspB transcription and increase both FT survival and recovery, but the physiological mechanism for this fitness improvement remains unknown.
EVOLUTIONARY biologists have long been interested in elucidating the genetic bases of adaptation to particular environments, including especially those environments that are novel or stressful to the organism. Evolution experiments using bacteria and other microorganisms (Elena and Lenski 2003; Poon and Chao 2005; Riehle et al. 2005; Herring et al. 2006; Schoustra et al. 2006; Velicer et al. 2006) offer a powerful context for studying the genetics of evolutionary adaptation, because one can couple changes in phenotypic traits, including fitness and its components, with specific mutations. In these studies, it is of interest to know whether independent populations, when confronted with the same environmental challenges, will evolve along parallel or divergent paths. The convergence of multiple evolving lines on similar phenotypes provides a strong indication that the changes are adaptive as opposed to the product of random genetic drift (Bull et al. 1997; Ferea et al. 1999;Wichman et al. 1999; Cooper et al. 2001, 2003; Colosimo et al. 2005; Wood et al. 2005; Pelosi et al. 2006; Woods et al. 2006).
Previous studies on evolutionary adaptation to stressful environments have focused on how known stressresponsive genes evolve (Riehle et al. 2001; De Visser et al. 2004). However, relatively few genes have been identified that are known to be important for adaptation to freeze-thaw (FT) stress, especially in mesophilic organisms such as Escherichia coli. When populations of E. coli are subjected to repeated FT cycles, no survivors remain in the population after 40 cycles (Sleight et al. 2006). To study how such populations may genetically adapt to these conditions over evolutionary time, 15 populations evolved under a freeze-thaw-growth (FTG) regime, where the growth phase allows for the selection of cells that can both survive and recover from the FT stress (Sleight and Lenski 2007). Here, we examine the genetic basis of evolutionary adaptation that occurred in these 15 FTG-evolved populations. The evolved lines achieved large increases in fitness relative to their progenitors when competed under the FTG regime, and these gains resulted from both improved survival after the FT cycle and faster recovery to initiate exponential growth after thawing (Sleight and Lenski 2007). This shorter lag phase is specific to recovery after freezing and thawing and not some more general improvement in recovery of growth following stationary phase per se. Thus, it is of interest to identify and characterize the genetic changes responsible for these adaptations to the FTG regime.
Various approaches have been used to find beneficial mutations substituted in bacterial evolution experiments (Treves et al. …