Characterization of A Ribose Metabolism Pathway in Bacteroides thetaiotaomicron and New Insights into the Nutrients Degraded by this Bacterium

Abstract

Bacteria of the Bacteroidetes phylum, dominant members within the gut microbiota, devote large genomic capacity towards nutrient acquisition via gene clusters termed polysaccharide utilization loci (PULs). The model organism, Bacteroides thetaiotaomicron (Bt) contains 88 PULs that target complex polysaccharides of host- microbial- or dietary origin. However, many PULs remain uncharacterized in terms of cognate substrate, enzyme functionality, and regulation. I have expanded the known substrates targeted through characterizing the ribose utilization system (rus) PUL in Bt. I created gene deletions based on predicted functionality within rus. Using these strains allowed for in vitro characterization of the substrates (e.g. ribose, nucleosides and RNA) that are catabolized through this PUL. The ability to access these nutrients confers a competitive advantage in vivo on a fiber-rich diet containing nucleosides. Additionally, through biochemical and in vivo studies I have connected the actions of a genomically unlinked nucleoside phosphorylase (BT4554) and the rus ribokinases (RusK1/K2). Determining that these two enzymes work together by BT4554 cleaving nucleosides which produces ribose-1-phosphate (R1P), which is subsequently phosphorylated by RusK1/K2 yielding ribose-1,5-bisphosphate (PRibP). Further, RusK2 accepts ribose-5-phosphate (R5P) as a substrate and synthesizes PRibP by phosphorylating the 1’C position. The functions displayed by RusK1 and RusK2 are the first described in eubacteria generating PRibP from R1P or R5P, and represents new metabolism in Bt. Further, the ability of Bt to sense ribose transcriptionally alter genes located within other PULs and loci. Contrastingly, to the rus PUL, mucin-O¬-glycan (MOG) PULs are strongly upregulated in vivo on a fiber-free diet (FF diet); a condition where Bt relies on host-derived glycans for growth. This FF diet resembles Westernized human diets that have been implicated in inflammatory bowel disorders (IBD) leading to colitis by bacteria eroding the host mucosa. By deleting MOG-responsive, sulfatase-encoding PULs in Bt as single PUL deletions and sequentially, (up to a strain lacking 10 PULs), I abrogated growth of Bt on MOG. This approach has assisted in narrowing the gene-encoded functions responsible for disease. Additionally, using a transposon mutagenesis screen, I was able to discover a Bt-specific T-cell epitope recognized in vivo during disease. The expression of this epitope is affected both by glucose and salt concentrations, demonstrating even more the interesting and largely unknown regulatory strategies employed by Bt. The regulatory network in Bt is complicated, with each PUL encoding its own regulatory protein (ECF-σ/anti-σ proteins, hybrid two-component systems, etc.). Additionally, Bt encodes 22 ECF-σ proteins as well as 4 LacI-type regulators not associated with known metabolic loci, making them orphan regulatory proteins. I have deleted most of these genes, resulting in discovery of a single ECF-σ gene, BT2492, which when deleted, reduces growth on 12 of the polysaccharides Bt degrades. Further, two LacI deletion strains result in drastically improved growth on normally low-priority monosaccharides. Lastly, as suggested by in vitro RNAseq data of ribose growth, the presence of ribose affects priority of other nutrients. This phenomenon extends to other simple sugars as arabinose and xylose RNAseq data reveal that they also exert changes in gene expression for loci not associated with their catabolism, including orphan ECF-σ factors. Together these data point to a complex regulatory cascade through a multi-faceted system involving PUL-encoded activators, trans-encoded proteins, and sugar-dependent prioritization through these mechanisms.