B. thetaiotaomicron is a versatile glycan foraging bacterium of the human gut microbiota. B. thetaiotaomicron can grow on a wide range of plant polysaccharides, and shows a high degree of nutritional flexibility to adapt to the substrates available in the gut (Koropatkin et al., 2008). The genome analysis of B. thetaiotaomicron revealed a comprehensive carbohydrate utilization system called the ‘Sus’ (starch utilization system or sequestration system) which is used to degrade and utilize starch. The genes encoding the proteins of the Sus system are organized into gene clusters called polysaccharide utilization loci (PULs). A minimum single unit PUL may possess genes encoding a set of enzymes that degrade a specific form of glycan substrate or closely related group of glycans, an outer membrane-associated substrate binding protein, and a substrate-specific receptor (Martens et al., 2011). The Sus locus typically consists of a cluster of 8 genes, susRABCDEFG, each encoding the components of a multi-protein complex that forms a cell-associated apparatus which binds, cleaves, and transports oligomeric starch into the intracellular regions where the imported substrates are enzymatically degraded (Figure 1.6) (Martens et al., 2009). SusC is an outer membrane- spanning β-barrel TonB-dependent receptor family that carries out a high affinity binding and the intake of substrate into the periplasmic region using the proton motive force (Martens et al., 2009). In general, active transport system is required especially when the substrates are poorly permeable through porins (e.g. large oligosaccharides). SusDEFG are lipoproteins containing bacterial signal peptidase II recognition motif (Marten et al., 2009). The concerted efforts of SusCDEF mediate
substrate binding close to the cell surface (Marten et al., 2009). SusC and SusD directly interact with each other, and are considered as a minimum unit of starch binding complex; disruptions in SusC and SusD completely abolished starch binding (Cho and Salyers, 2001). SusC and SusD are responsible for approximately 60% of starch binding observed in wildtype B. thetaiotaomicron (Martens et al 2009). SusD mediates starch and malto-oligosaccharide binding via a single starch-binding site (Koropatkin et al., 2008). The analysis of SusD crystal structure revealed that the protein has a unique α-helical fold which recognizes the 3D helical structure of starch molecules instead of the relative spatial arrangement of individual glucose residues (Koropatkin et al., 2008). SusE and SusF are surface-exposed lipoproteins containing multiple starch-binding domains which facilitate a high- affinity substrate binding by B. thetaiotaomicron (Cameron et al., 2012). Although not essential for growth, the inclusion of SusE in SusCDE complex increased the starch binding affinity to more than 80%, while the inclusion of SusF in SusCEDF complex completely restored the starch binding affinity
Figure 1.6 Schematic diagram of starch utilization system (Sus) of B. thetaiotaomicron (Martens et al., 2009). No copyright permission is required for the use of this figure.
to 100% (Martens et al., 2009, Shipman et al., 2000). SusG is an endo-acting starch-degrading enzyme tethered to the outer membrane protein (Shipman et al., 1999). Sharing a high sequence similarity to α-amylase, the starch-degrading ability of SusG is essential for the organism’s growth on long chain starch molecules (Shipman et al., 1999). It is speculated that the endo-acting SusG may introduce internal cuts within the bound starch, generating shorter oligosaccharides that can be transported through SusC porins (Martens et al., 2009). Once in the periplasm, glycoside hydrolases (GHs) SusA and SusB degrade the oligosaccharides into simpler sugars before their transport into the cytosol. SusA is a neopullulanase homologue which is a typical starch-hydrolysing protein (Kitamura et al., 2008). SusB is an α-glycosidase belonging to GH family 97 (Kitamura et al., 2008). SusR is a transcriptional level controller for SusABCDEFG expression (Martens et al., 2009). While SusR is expressed constitutively in wildtype, increasing the expression of SusR increased the expression of SusA, B, and C as well as the overall growth rate of B. thetaiotaomicron on starch (DElla et al., 1996).
B. thetaiotaomicron and other Bacteroides spp. use the Sus-like systems to degrade a wide range of plant cell wall polysaccharides and the host-derived mucin O-glycans. The Sus paradigm is a typical example of a highly conserved carbohydrate utilization system showing a diversity in saccharolytic ability due to the variations in the catalytic components of the multi-protein complex. For example, the transcriptomic profiling of B. thetaiotaomicron grown on pectic glycans revealed that at least 10 PULs showed a significantly induced expression, indicating the enzymes encoded by pectin-specific PULs were activated in response to the availability of pectin substrates (Martens et al., 2011). Unlike B. thetaiotaomicron that cannot degrade hemicellulose, the PULs of B. ovatus were enriched with genes enabling the metabolism of all types of hemicelluloses (Martens et al., 2011). B. ovatus PULs contained at least a pair of genes encoding homologs of B. thetaiotaomicronsusC/D and various enzyme families implicated in degrading xylan, xyloglucan and galactomannan, which showed increased expressions in the presence of hemicellulose (Martens et al., 2011). In Bacteroides cellulosilyticus, the expression of sus gene homologues was significantly upregulated by the consumption of high plant polysaccharide diet (McNulty et al., 2013). Although some strains of B.
cellulolyticus were capable of degrading cellulose, their preferred source of carbohydrate was xylan, a major component of hemicelluloses. Among the most highly expressed carbohydrate-degrading enzymes of B. cellulolyticus in response to the plant polysaccharide-rich diet, many belonged to the enzyme families required for degrading xylan and pectin (McNulty et al., 2013). B. xylanisolvens showed a higher xylanolytic activity than B. ovatus, and was able to degrade xylans from diverse sources (Mirande et al., 2010). B. xylanisolvens genome also contained over 100 genes coding for enzymes potentially involved in the pectin degradation (Despres et al., 2016). The genes coding for pectate lyases and polygalacturonases targeting the pectin backbone structure were found in 5 out of 6 PULs which showed a significant overexpression when B. xylanisolvens was grown using citrus and apple pectin (Despres et al., 2016).
The homologues of Sus system genes were also found in the rumen/gut starch- and hemicelluloses- degrading species of Prevotella (previously classified as Bacteroides), indicating a deep evolutionary root and the distribution of the system among the members of Bacteroidetes occurring through an extensive gene transfer and gene duplications (Dodd et al., 2010).