Abstract 2420 Interactions between the TonB dependent transporter of the starch utilization system in Bacteroides thetaiotaomicron and two different TonB proteins and progress towards a long-sought structure of the SusC/D complex

Bacteroides thetaiotaomicron (B. theta) is a prominent member of the human gut which specializes in capturing a diverse array of carbohydrates through systems involving TonB dependent transporters (TBDTs). TBDTs interact with an inner membrane TonB protein to facilitate transport of substrate from outside the cell into the periplasm. One such system is the well-studied Starch Utilization System (Sus) which is comprised of 8 proteins dedicated to the import of starch across the outer membrane and subsequent breakdown into glucose. Membrane transport is mediated by the TBDT SusC along with a set of outer-membrane lipoproteins (SusDEFG) that capture starch chains and cleave them into smaller oligosaccharides. SusC-like TBDTs are highly conserved across other polysaccharide utilization systems but much of the mechanism of transport for SusC and SusC-like TBDTs remains uncharacterized. SusC-like TBDTs include several unique features that separate them from more classically understood TBDTs including a unique structural domain at the N-terminus, which may facilitate pairing between the transporter and TonB. In addition, there are 11 putative tonB genes encoded in B. theta. The deletion of tonB4 results in the largest growth deficit on starch but this deletion fails to abolish growth entirely, suggesting at least one other putative tonB gene that can also facilitate starch transport through SusC. The goal of this work is elucidation of the interactions between TonBs and SusC from a physiological and structural perspective. Quantitative proteomics done on TonB proteins from B. theta grown on glucose and maltose (which upregulates the sus genes) showed that when tonB4 is deleted, the abundance of TonB6 is drastically increased from nearly undetectable levels. This suggests TonB6 can act as a backup to TonB4 for SusC function. In addition, we performed chemical crosslinking using formaldehyde in live cells grown on potato starch followed by affinity purification of SusCD and then mass spectrometry to determine which proteins were captured with it. This preliminary data revealed TonB4 and TonB6 were captured, suggesting both are capable of powering SusCD. We have also purified a native complex of SusCD via a 6x his tag on SusD using affinity and size exclusion chromatography resulting in a highly pure complex for cryo-electron microscopy which will serve to elucidate the detailed structure of the unique features of this TBDT. This early work suggests that TonB4 and TonB6 facilitate SusC function and isolation of the SusCD complex will allow us to better understand how these interactions are mediated. Our future work will continue to address the relationship between TonB and SusC starch transport using structural, proteomic, and physiological techniques. This work is primarily supported by a Research Program Project grant (P01-HL149633 to N.M.K.) from the National Heart, Lung and Blood Institute (NHLBI) of the National Institutes of Health (NIH).