Abstract 2048 Targeting Histone H2A Variant Genes in Bdelloid Rotifers using sgRNA/Cas9 CRISPR Complexes

Bacteroides are vital members of the human gut microbial community that are primarily responsible for the utilization of polysaccharides.Bacteroides thetaiotaomicron (B.theta) is a model organism for the Bacteroides genus and transport of degraded oligosaccharides through TonB-dependent transporters (TBDT).These outer membrane transporters are powered by an inner membrane energizing complex composed of the TonB, ExbB, and ExbD proteins.Previous research has identified 11 homologous TonB proteins encoded in the B. theta genome.Analysis of B. theta strains containing single tonB gene deletions suggests that TonB4 is the primary protein used in the energizing complex associated with polysaccharide transporters but TonB6 has a redundant function.To further investigate the roles of TonB4 and TonB6, this work seeks to generate multi-deletion strains expressing only the TonB4 or TonB6 protein and a double deletion strain expressing all TonB proteins with the exception of TonB4 and TonB6.To make strains expressing only TonB4 or TonB6, we aim to conduct deletions of the other 9 tonB homologs and have successfully deleted tonB1, 2, 3, 5, 8, 9, 10, and 11 leaving tonB 4, 6, and 7. Previous work to produce a tonB4 and tonB6 double deletion strain have not been successful, perhaps because the double deletion results in a lethal phenotype.To address this limitation, we have produced tonB4 and tonB6 single deletion strains with the corresponding protein expressed under an inducible promoter at a third genomic site, allowing for the second tonB gene to be deleted.Though a double deletion strain has not yet been isolated, screening data suggests this is a viable approach.Generation of these strains will provide additional tools for understanding the role of TonB in polysaccharide utilization with the goal of differentiating between the energizing complexes in beneficial bacteria such as B. theta and closely related pathogenic species including B. fragilis.