Mycoplasmas under experimental antimicrobial selection: The unpredicted contribution of horizontal chromosomal transfer

Horizontal Gene Transfer was long thought to be marginal in Mycoplasma a large group of wall-less bacteria often portrayed as minimal cells because of their reduced genomes (ca. 0.5 to 2.0 Mb) and their limited metabolic pathways. This view was recently challenged by the discovery of conjugative exchanges of large chromosomal fragments that equally affected all parts of the chromosome via an unconventional mechanism, so that the whole mycoplasma genome is potentially mobile. By combining next generation sequencing to classical mating and evolutionary experiments, the current study further explored the contribution and impact of this phenomenon on mycoplasma evolution and adaptation using the fluoroquinolone enrofloxacin (Enro), for selective pressure and the ruminant pathogen Mycoplasma agalactiae, as a model organism. For this purpose, we generated isogenic lineages that displayed different combination of spontaneous mutations in Enro target genes (gyrA, gyrB, parC and parE) in association to gradual level of resistance to Enro. We then tested whether these mutations can be acquired by a susceptible population via conjugative chromosomal transfer knowing that, in our model organism, the 4 target genes are scattered in three distinct and distant loci. Our data show that under antibiotic selective pressure, the time scale of the mutational pathway leading to high-level of Enro resistance can be readily compressed into a single conjugative step, in which several EnroR alleles were transferred from resistant to susceptible mycoplasma cells. In addition to acting as an accelerator for antimicrobial dissemination, mycoplasma chromosomal transfer reshuffled genomes beyond expectations and created a mosaic of resistant sub-populations with unpredicted and unrelated features. Our findings provide insights into the process that may drive evolution and adaptability of several pathogenic Mycoplasma spp. via an unconventional conjugative mechanism.
Author summary Genome downsizing is often viewed as a degenerative process of evolution. Such erosion has left current mycoplasmas with a minimal genome: for some species its size barely exceeds the amount of information needed for sustaining autonomous life. Despite such limitations, these simple bacteria showcase a baffling capacity for adaptation to complex environments such as that provided by the animal host. By using the enrofloxacin antibiotic as selective pressure, we performed a genome scale analysis of macro- and micro-events leading to antimicrobial resistance in mycoplasmas. Sexually competent cells were found to shortcut this process by using an unconventional mechanism of chromosomal transfer driving massive exchanges of DNA materials. Remarkably, this powerful mechanism was associated with a profound genomic reorganization that reshuffled parental features and created mosaicism. This finding emphasizes the extraordinary adaptability of some pathogenic Mycoplasma spp. and provides major insights into the processes that contribute to shaping the evolution of their minimal genome. While unconventional conjugative mechanisms are being documented in more complex bacteria, the reduced mycoplasma genome may provide a simplified model to study mosaicism and its role in bacterial evolution.