When trying to construct an accurate model of PUL-mediated glycan harvest, determining the cellular location of the PUL encoded glycosidic-linkage breaking enzymes becomes as important as understanding the enzymes' specificity (). Analysis of the predicted cellular locations of the glycoside hydrolases (GH) from the four efficient inulin-using Bacteroides fructan PULs studied suggest that all four Bacteroides species have at least one enzyme that is likely to be an outer membrane lipoprotein, and thus may be cell surface located. Studies with the only two Sus-like systems characterized to date (the B. thetaiotaomicron
starch and levan systems) have shown that the surface located enzyme from each of these cleaves its polymeric substrate in an endo-like fashion, producing oligosaccharides that are transported into the cell via the SusC porin.1,4,8
In B. caccae
and B. ovatus
the putative surface located enzymes are GH91s, a family known to possess endo-inulinase activity.10
However, in B. fragilis
and B. uniformis
the putative surface enzyme is a GH32 that is closely related to B. thetaiotaomicron
enzymes that we have shown to be non-linkage specific exo-fructosidases (see ref. 1
, Fig. S6; BF3177 and BACUNI_01159), which leads to the question: are these enzymes endo-acting inulinases or are they actually producing fructose extracellularly? The latter possibility seems unlikely as the production of a monosaccharide would negate the requirement for any periplasmic glycoside hydrolases and a SusC ortholog for transport of oligosaccharides.
B. vulgatus is the only sequenced Bacteroides to date that lacks susC- or susD-like genes within its fructan PUL and cannot use inulin or levan. However, B. vulgatus is able to grow well on short-chain β2-1 fructo-oligosaccharides (FOS; Sonnenburg J, unpublished). Intriguingly, B. vulgatus contains only a single fructan-degrading enzyme (a GH32; BVU_1663), which is predicted to be an outer membrane located lipoprotein and may therefore be surface located. Comparison of the sequence of BVU_1663 with the B. thetaiotaomicron GH32s reveals it is most closely related to BT1765, an exo-fructosidase enzyme that displays a strong preference for short chain fructo-oligosaccharides over polymeric fructans. If BVU_1633 displays a similar substrate size preference to BT1765, this may explain the ability of B. vulgatus to utilize FOS, but not inulin. The enzyme's predicted location would suggest that the production of fructose occurs extracellularly. Thus, the likely mechanism of FOS utilization by this bacterium provides a ready explanation for its lack of genes encoding SusC and SusD homologs, as it does not require such an elaborate system to import fructose across the outer membrane. It remains to be determined if B. vulgatus has specialized in the use of FOS present in the diet, cross-feeds on short fructans generated extracellularly by other long-chain fructan-users, or has lost the ability to use long-chain fructans for another reason.
The data described for B. vulgatus
supports the view that SusC- and SusD-like proteins are required for utilization of polymeric fructans. However, it is currently unclear whether an extracellular endo-acting enzyme is also an absolute requirement. With respect to the efficient polymeric inulin users, an alternative possibility is that some currently uncharacterized Bacteroides species lack an extracellular fructan-utilizing enzyme altogether. In this case the inulin chains would be threaded through the SusC porin without being cleaved outside the cell and all degradation would occur in the periplasm. Although this is clearly not the case in B. thetaiotaomicron
's levan utilization system,1
it may be possible with inulin. This polysaccharide has a relatively short average degree of polymerization of only ~25 fructose units (for Chicory inulin) and may not require external processing by the cell for transport through the outer membrane via the SusC/D system.11
We are currently working to determine whether SusC/D systems are always paired with extracellular endo-polysaccharidases, or whether a sub-class of PULs work in the absence of extracellular oligosaccharide generation.
The apparent redundant specificity and periplasmic location of two of the B. thetaiotaomicron
GH32 enzymes, BT3082 and BT1759, provides another unanswered question. The redundancy contrasts with the prototypic Sus that relies upon a single periplasmic glucosidase, SusB, which is capable of cleaving four different α-glucosidic linkages.12
One possibility is that one of the enzymes is actually optimized to deal with more branched form of fructans such as graminins, which are mixed β2-1/2-6 linked fructans that are found mainly in grass species such as wheat and barley.13
While it is not known if B. thetaiotaomicron
can actually utilize these highly branched substrates, it would seem an obvious rationale for the duplicity of apparently similar enzyme specificities. Indeed, the presence of four predicted fructan-processing enzymes encoded within the fructan PULs of several of the other inulin-using Bacteroides species strengthens the idea that fructan use in these strains is more complex than simply β2-1 or β2-6. Interestingly, B. fragilis
contains only two GH32s, suggesting it specifically targets inulin and that branched oligosaccharides are precluded from entry into the periplasm by the specificity of the B. fragilis
SusD. Activity screening with branched forms of fructan, combined with biochemical and gene knockout studies, should enable us to dissect the role that some of these apparently redundant enzymes play.