Genetic modification of , a non-pathogenic CTG species, reveals function.

is closely related to important pathogenic species, especially and but it has been rarely isolated from humans. For this reason, through comparative studies, it could be a powerful model to understand the genetic underpinnings of the pathogenicity of species. Here, we generated a cohesive assembly of the genome and developed genetic engineering tools that will facilitate studying this species at a molecular level. We used a combination of short and long-read sequencing to build a polished genomic draft composed of 14 Mbp, 45 contigs and close to 5700 genes. This assembly represents a substantial improvement from the currently available sequences that are composed of thousands of contigs. Genomic comparison with and revealed a substantial reduction in the total number of genes in . However, gene loss seems not to be associated to the avirulence of this species given that most genes that have been previously associated with pathogenicity were also present in . To be able to edit the genome of we generated a set of triple auxotrophic strains so that gene deletions can be performed similarly to what has been routinely done in pathogenic species. As a proof of concept, we generated gene knockouts of a gene that encodes a transcription factor that is essential for filamentation and biofilm formation in and . Characterization of these mutants showed that Efg1 also plays a role in biofilm formation and filamentous growth in , but it seems to be a repressor of filamentation in this species. The genome assembly and auxotrophic mutants developed here are a key step forward to start using for comparative and evolutionary studies at a molecular level.