Abstract 1797 Analyzing growth patterns of ExbD gene deletions bt3920 and bt3921 in Bacteroides thetaiotaomicron

Polysaccharides are a common source of energy through digestion in the human gut microbiota. Without the microbiota, degradation of polysaccharides into viable energy would be nearly impossible for humans. In order to improve and manipulate this degradation, a better understanding of the bacterial cells that live within the microbiota is imperative. Bacteroides thetaiotaomicron (B. theta) is one of the common bacteria that live within the human gut and are an important contributor to processing polysaccharides. B. theta uses a polysaccharide utilization loci (PUL), TonB-dependent transporter, TonB protein, and ExbB and ExbD complex to transport polysaccharides into their intermembrane space. This is possible by the TonB protein pulling on the transporter through the power of the proton motive force generated from the rotational motion of the ExbB and ExbD complex. To further understand this mechanism and how each component plays a part in successful transportation of polysaccharides, we are interested in deleting the ExbD homologs bt3920 and bt3921 separately and characterizing the impact of these deletions on polysaccharide transport. bt3920 and bt3921 are predicted ExbD homologs. Primers were created from the genome region flanking each gene and were then amplified and inserted into a pExchange plasmid. Bacterial recombination was then used to insert the genomic region containing the gene deletion into B. theta. Construction of the pExchange plasmid containing a genetic region without bt3920 or bt3921 was successful and evident through gel electrophoresis and sequencing. The Δbt3920 pExchange plasmid was then conjugated into B. theta. The conjugated B.theta was confirmed to be the final deletion strain and growth on only glucose aligns with wildtype growth. Further directions of this research will be to construct a bt3921 deletion strain into B. theta and then measure how different polysaccharide sources effect the growth pattern of B. theta Δ3920 and Δ3921.