Measurement of abiotic adaptation of bacterial populations
The abiotic adaptation of bacteria from each low-temperature microcosm was determined by measuring growth performance in the absence of phages. To do so, bacterial populations in coevolution microcosms (that included both bacteria and phages) were first isolated by Virkon treatment. Specifically, KB medium with 0.375% of the disinfectant Virkon (Antec International, Sudbury, England) was prepared; and 60 µL of each culture was added to 6 mL of the Virkon-supplemented medium and left for 24 h at 28°C. This procedure left bacterial viable and completely phage free; then 60 µL of each Virkon-treated culture was added to 6 mL of fresh KB and grown for 24 h to give a phage-free and Virkon-free culture (Morganet al. 2005). The potential presence of phages in Virkon-treated cultures was tested for by spotting cultures onto semi-solid agar seeded with ancestral P. fluorescens strain and incubated at 28°C for 24 h; plaques would indicate the presence of phages. Bacteria from all except one coevolution lines were successfully rescued from phages; and the one exception was a BP+IBP microcosm where bacterial population failed to revive and thus was excluded in the measurement of abiotic adaptation.
Each bacterial population from the low-temperature microcosms, as well as the ancestral strain, was acclimated at 10°C for 48 h, 1% of which was transferred into fresh medium and incubated for another 48 h. Optical densities of resultant cultures were measured (mOD600), as a surrogate of bacterial density (three replicates for each assay and the mean value used in subsequent analyses).