Determinants of raffinose family oligosaccharide use in Bacteroides species.

ABSTRACT

Bacteroides species are successful colonizers of the human gut and can utilize a wide variety of complex polysaccharides and oligosaccharides that are indigestible by the host. To do this, they use enzymes encoded in Polysaccharide Utilization Loci (PULs). While recent work has uncovered the PULs required for use of some polysaccharides, how Bacteroides utilize smaller oligosaccharides is less well studied. Raffinose family oligosaccharides (RFOs) are abundant in plants, especially legumes, and consist of variable units of galactose linked by α-1,6 bonds to a sucrose (glucose α-1-ß-2 fructose) moiety. Previous work showed that an α-galactosidase, BT1871, is required for RFO utilization in Bacteroides thetaiotaomicron. Here, we identify two different types of mutations that increase BT1871 mRNA levels and improve B. thetaiotaomicron growth on RFOs. First, a novel spontaneous duplication of BT1872 and BT1871 places these genes under control of a ribosomal promoter, driving high BT1871 transcription. Second, nonsense mutations in a gene encoding the PUL24 anti-sigma factor likewise increase BT1871 transcription. We then show that hydrolases from PUL22 work together with BT1871 to break down the sucrose moiety of RFOs and determine that the master regulator of carbohydrate utilization (BT4338) plays a role in RFO utilization in B. thetaiotaomicron. Examining the genomes of other Bacteroides species, we found homologs of BT1871 in subset and show that representative strains of species containing a BT1871 homolog grew better on melibiose than species that lack a BT1871 homolog. Altogether, our findings shed light on how an important gut commensal utilizes an abundant dietary oligosaccharide.