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. IMPORTANCE 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.

ABSTRACT Lymphatic filariasis is a devastating disease caused by filarial nematode roundworms, which contain obligate Wolbachia endosymbionts. Here, we assembled the genome of wBp, the Wolbachia endosymbiont of the filarial nematode Brugia pahangi, from Illumina, Pacific Biosciences, and Oxford Nanopore data. The complete, circular genome is 1,072,967 bp.

Symbioses between animals and bacteria are ubiquitous. To better understand these relationships, it is essential to unravel how bacteria evolve to colonize hosts. Previously, we serially passaged the free-living bacterium, Shewanella oneidensis, through the digestive tracts of germ-free larval zebrafish (Danio rerio) to uncover the evolutionary changes involved in the initiation of a novel symbiosis with a vertebrate host. After 20 passages, we discovered an adaptive missense mutation in the mshL gene of the msh pilus operon, which improved host colonization, increased swimming motility, and reduced surface adhesion. In the present study, we have determined that this mutation was a loss-of-function mutation and found that it improved zebrafish colonization by augmenting S. oneidensis representation in the water column outside larvae through a reduced association with environmental surfaces. Additionally, we found that strains containing the mshL mutation were able to immigrate into host digestive tracts at higher rates per capita. However, mutant and evolved strains exhibited no evidence of a competitive advantage after colonizing hosts. Our results demonstrate that bacterial behaviors outside the host can play a dominant role in facilitating the onset of novel host associations.

RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an “Alternative Model” to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including virus, bacteria, fungi, and animals. The algorithm consistently identified a 5-methylcytosine at the central position of a GCU motif. However, it also identified a 5-methylcytosine in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this a frequent false prediction. In the absence of further validation, several published predictions of 5-methylcytosine in human coronavirus and human cerebral organoid RNA in a GCU context should be reconsidered.