Super-Resolution Imaging of Live Anaerobic Bacteria: Assembly and Dynamics of the B. Theta Starch Utilization System

Abstract

The human gut hosts trillions of bacteria that directly influence human health. The majority of these gut microbiota play an important role in nutrition by metabolizing host-indigestible complex glycans into short-chain fatty acids. Bacteroides thetaiotaomicron (Bt), a prominent bacterial symbiont in the distal gut, metabolizes over a dozen complex glycans using membrane-associated protein complexes. The Starch Utilization System (Sus) is the first recognized multi-protein complex in Bt cells that is essential for growth on starch. Bt-Sus uses eight proteins (SusRABCDEFG) to process starch; though, only SusD and SusG are vital for cellular proliferation in the presence of starch. SusCDEFG localize in the outer membrane and likely form a complex to facilitate starch binding, degradation and import. However, conventional biochemical methods have been unable to completely reveal the assembly and dynamics of these proteins in response to starch. We have applied single-molecule super-resolution imaging to characterize the Sus complex response to different sugars in live Bt cells under anaerobic conditions. HaloTag-labeled SusG was tracked concurrently with other fluorescent antibody-labeled Sus proteins. Our initial protein correlation studies demonstrated that simple sugars such as glucose or maltose do not induce Sus complex assembly. On the other hand, incubation of Bt cells with amylopectin, a constituent of starch, enhanced the colocalization of Sus proteins suggesting starch-mediated assembly of the Sus complex. Furthermore, we have used single-molecule tracking experiments to quantify Sus protein movement in the presence and absence of starch in live cells. We have discovered two distinct modes of SusG: a diffusing population and a stationary population. This latter group is mainly observed for cells on amylose-coated beads and is consistent with starch-bound SusG.