Abstract 1686 Characterizing the Role of TonB7 in Bacteroides thetaiotaomicron

Bacterial microorganisms are crucial for human health and digestion. The Bacteroides are prevalent members of the gut microbial community that are essential for polysaccharide degradation and formation of a healthy gut microbiome. In the gram-negative model organism Bacteroides thetaiotaomicron (B. theta), specialized membrane proteins known as TonB-Dependent Transporters (TBDTs) facilitate the movement of complex polysaccharides into the cell. This transport is energized by TonB proteins in the inner membrane. Previous research on the 11 identified TonB proteins in B. theta involved characterizing their role in polysaccharide utilization through gene deletions. This research showed that although the TonB7 protein (BT3192) was not present in the membrane fraction of glucose or maltose grown cells, deletion of this gene produced a lag in bacterial growth that was not rescued by complementation. This suggests that removing TonB7 from the genome disrupts some essential process(es) other than TonB interaction with the polysaccharide transporter. To achieve a ΔTonB7 strain without this phenotype, the deletion scheme was redesigned to target two highly conserved portions of the gene that are predicted to interact with the TBDT: the entire C-terminal domain (CTD) and a beta-sheet within that domain. These regional deletion strains were grown on glucose to assess growth changes compared to wild-type and ΔTonB7 B. theta. The lag phenotype was absent in both ΔCTD and Δβ-sheet TonB7 strains suggesting we have been able to construct a deletion that eliminates interactions with the TBDT but does not disrupt other processes in the bacteria. Additional growth assays on starch will inform TonB7's role in polysaccharide transport. Multi-deletion strains will also be constructed using the ΔTonB7 regional deletions to explore TBDT specificity for the 11 TonB proteins. Comparison of the B. theta TonB proteins to those encoded by other Bacteroides species suggests that TonB7 is found only in a limited number of species closely related to B. theta while TonB4 (BT2059) is highly conserved across the Bacteroides. Our novel deletion construct allowing for disruption of TonB7 function without inducing a lag on glucose enables further investigation of TonB redundancy and TBDT specificity.