Identifying essential genes in using a saturated transposon library.

The unique epibiotic-parasitic relationship between type strain TM7x, a member of the newly identified candidate phyla radiation, now referred to as , and its basibiont, strain XH001 (formerly ), requires more powerful genetic tools for a deeper understanding of the genetic underpinnings that mediate their obligate relationship. Previous studies have mainly characterized the genomic landscape of XH001 during or post-TM7x infection through comparative genomic or transcriptomic analyses, followed by phenotypic analysis. Comprehensive genetic dissection of the pair is currently cumbersome due to the lack of robust genetic tools in TM7x. However, basic genetic tools are available for XH001, and this study expands the current genetic toolset by developing high-throughput transposon insertion sequencing (Tn-seq). Tn-seq was employed to screen for essential genes in XH001 under laboratory conditions. A highly saturated Tn-seq library was generated with nearly 660,000 unique insertion mutations, averaging one insertion every two-three nucleotides. A total of 203 genes comprising 10.5% of the XH001 genome were identified as putatively essential.IMPORTANCE strain XH001, an early colonizer of the oral multispecies biofilm (dental plaque), forms a unique epibiotic-parasitic relationship with type strain TM7x, a member of the newly identified (formerly candidate phyla radiation). Achieving a mechanistic understanding of their relationship requires practical genetic tools for dissecting the roles played by different genetic mediators and shedding light on how their interspecies interaction may affect dynamics in the oral microbiome. In this study, we developed a high-throughput mutagenesis technique, Tn-seq, in XH001. The constructed Tn-seq library enabled the identification of putatively essential genes in XH001, revealing growth requirements under laboratory conditions. This library can be leveraged in future studies to elucidate TM7x's dependence on XH001 at the molecular level.