Experimental Adaptation of a Free-Living Bacterium to the Zebrafish Digestive Tract

Animals have coexisted with an omnipresent and diverse array of bacteria for the entirety of their evolutionary history. As a result, symbioses between animals and bacteria are ubiquitous and can range from mutualism to parasitism. In particular, countless studies have demonstrated the pivotal role that bacteria residing in animal digestive tracts can play in determining animal health and well-being. However, it is still unknown how bacteria evolve the ability colonize animals. Due to the dramatic impacts that animals and bacteria can have on one another’s fitness, it is imperative to understand how symbioses between bacteria and their animal hosts originate. Therefore, to elucidate how bacteria evolve novel associations with vertebrate hosts, I serially passaged six replicate populations of a bacterial species with no prior known host associations (Shewanella oneidensis) through the digestive tracts of a model vertebrate, zebrafish (Danio rerio). After 20 passages through the digestive tracts of groups of larval zebrafish that were derived bacteria free (amounting to approximately 200 bacterial generations), I observed that all six replicate populations evolved to outcompete their unpassaged ancestor in terms of their ability to colonize larval guts. I subsequently sequenced the genomes of four evolved S. oneidensis isolates from each replicate population and found that their competitive advantage stemmed from two distinct classes of mutations that occurred in a mannose sensitive hemagglutinin pilus operon as well as in genes with putative diguanylate cyclase and phosphodiesterase domains. Both types of mutations enhanced bacterial motility, which was associated with increased representation in the aqueous portion of my experimental system and more efficient per capita immigration into zebrafish guts relative to the ancestral S. oneidensis reference strain. These increases in motility, were consistent with the behavior of a closely-related Shewanella species (Shewanella sp. ZOR0012) that has recently been isolated from the zebrafish digestive tract implying that my evolved isolates may be pursuing a similar adaptive trajectory to the one taken by this host-associated species. My results suggest that a non-host-associated microorganism can rapidly improve its ability to colonize hosts, and this study is the first to capture the early adaptive steps necessary to facilitate this transition

Phenotypic Parallelism during Experimental Adaptation of a Free-Living Bacterium to the Zebrafish Gut

Although animals encounter many bacterial species throughout their lives, only a subset colonize vertebrate digestive tracts, and these bacteria can profoundly influence the health and development of their animal hosts. We used experimental evolution to study a free-living bacterium as it adapts to a novel vertebrate host by serially passaging replicate populations of Shewanella oneidensis through the intestines of larval zebrafish (Danio rerio). Our results demonstrate that adaptation to the zebrafish gut is complex, with multiple evolutionary pathways capable of improving colonization, but that motility plays an important role during the onset of host association.Although animals encounter a plethora of bacterial species throughout their lives, only a subset colonize vertebrate digestive tracts, and these bacteria can profoundly influence the health and development of their animal hosts. However, our understanding of how bacteria initiate symbioses with animal hosts remains underexplored, and this process is central to the assembly and function of gut bacterial communities. Therefore, we used experimental evolution to study a free-living bacterium as it adapts to a novel vertebrate host by serially passaging replicate populations of Shewanella oneidensis through the intestines of larval zebrafish (Danio rerio). After approximately 200 bacterial generations, isolates from evolved populations improved their ability to colonize larval zebrafish during competition against their unpassaged ancestor. Genome sequencing revealed unique sets of mutations in the two evolved isolates exhibiting the highest mean competitive fitness. One isolate exhibited increased swimming motility and decreased biofilm formation compared to the ancestor, and we identified a missense mutation in the mannose-sensitive hemagglutinin pilus operon that is sufficient to increase fitness and reproduce these phenotypes. The second isolate exhibited enhanced swimming motility but unchanged biofilm formation, and here the genetic basis for adaptation is less clear. These parallel enhancements in motility and fitness resemble the behavior of a closely related Shewanella strain previously isolated from larval zebrafish and suggest phenotypic convergence with this isolate. Our results demonstrate that adaptation to the zebrafish gut is complex, with multiple evolutionary pathways capable of improving colonization, but that motility plays an important role during the onset of host association