Plasmid transmission dynamics and evolution of partner quality in a natural population of <i>Rhizobium leguminosarum</i>

Many bacterial traits important to host-microbe symbiosis are determined by genes carried on extrachromosomal replicons, such as plasmids, chromids, and integrative and conjugative elements. Multiple such replicons often coexist within a single cell and, due to horizontal mobility, have patterns of variation and evolutionary histories that are distinct from each other and from the bacterial chromosome. In nitrogen-fixing <i>Rhizobium</i>, genes carried on multiple plasmids make up a third of the genome, are necessary for the formation of symbiosis, and underlie bacterial traits, including host plant benefits. Thus, the genomics and transmission of plasmids in <i>Rhizobium</i> underlie the ecology and evolution of this important model symbiont. Here, we leverage a natural population of clover-associated <i>Rhizobium</i> in which partner quality has declined in response to long-term nitrogen fertilization. We use 62 novel, reference-quality genomes to characterize 256 replicons in the plasmidome and study their genomics and transmission patterns. We find that, of the four most frequent plasmid types, two (types II and III) have more stable size, larger core genomes, and track the chromosomal phylogeny (display more vertical transmission), while others (type I and type IV, or symbiosis plasmid, pSym) vary substantially in size and shared gene content and have phylogenies consistent with frequent horizontal transmission. We also find differentiation in pSym subtypes driven by long-term nitrogen fertilization. Our results highlight the variation in plasmid transmission dynamics within a single symbiont and implicate plasmid horizontal transmission in the rapid evolution of partner quality.IMPORTANCEUnderstanding how bacterial genes move through natural populations is critical for understanding how bacterial traits evolve. Nitrogen-fixing bacteria <i>Rhizobium leguminosarum</i> live in symbiosis with plants and are a model for studying plasmid transmission and how mobile genetic elements impact the evolution of bacteria and plants. Here, we characterize the genomes of a natural bacterial population, then use novel approaches to show that mechanisms of gene transmission vary across multiple plasmid types that coexist within <i>R. leguminosarum</i> cells. We find that changes in the frequency of specific pSym types are associated with the decline of symbiotic partner quality in strains isolated from environments undergoing long-term fertilization. These results underscore the importance of plasmid transmission and evolution in shaping ecosystem processes like nitrogen cycling via bacterial-plant symbiosis. Our study provides a framework for probing plasmid dynamics within natural bacterial populations and how plasmid transmission affects genetic diversity and ecological interactions in bacteria.