Genome dynamics and temperature adaptation during experimental evolution of obligate intracellular bacteria

Evolution experiments with free-living microbes have radically improved our understanding of genome evolution and how microorganisms adapt to different environments. Yet there is a paucity of such research focusing on strictly host-associated bacteria, even though they are so widespread in nature. Here, we used the Acanthamoeba symbiont Protochlamydia amoebophila, a distant relative of the human pathogen Chlamydia trachomatis and representative of a large group of ubiquitous protist-associated environmental chlamydiae, as a model to study how obligate intracellular symbionts made the pivotal evolutionary leap from protists to multicellular eukaryotes with respect to adaptation to elevated temperature. We established replicate populations under two temperatures (20 C, 30 C) for 510 generations (38 months). We then used a combination of infectivity assays and pooled whole-genome re-sequencing to identify any evolved phenotypes and the molecular basis of adaptation in these bacteria. Despite an overall reduction in infectivity of the symbionts that evolved at 30 C, symbionts were better adapted to infect at the temperature they were maintained in, be it ambient or elevated. We identified numerous nonsynonymous mutations and small indels (< 4 nt) in symbionts evolved at 30 C, with numerous variants persisting throughout multiple time points and reaching high frequencies. This suggests that many mutations may have been beneficial and played an adaptive role. Between the two temperature regimes the genes affected by mutations strongly differed with respect to their functional roles. Our results suggest that temperature adaptation was facilitated through attenuation of symbiont infectivity as a trade-off to reduce host cell burden.