Biochemical evidence that starch breakdown by Bacteroides thetaiotaomicron involves outer membrane starch-binding sites and periplasmic starch-degrading enzymes

Abstract

Bacteroides thetaiotaomicron can utilize amylose, amylopectin, and pullulan as sole sources of carbon and energy. The enzymes that degrade these polysaccharides were found to be primarily cell associated rather than extracellular. Although some activity was detected in extracellular fluid, this appeared to be the result of cell lysis. The cell-associated amylase, amylopectinase, and pullulanase activities partitioned similarly to the periplasmic marker, acid phosphatase, when cells were exposed to a cold-shock treatment. Two other enzymes associated with starch breakdown, at-glucosidase and maltase, appeared to be located in the cytoplasm. Intact cells of B. thetaiotaomicron were found to bind '4C-starch. Binding was probably mediated by a protein because it was saturable and was decreased by treatment of cells with proteinase K. Results of competition experiments showed that the starch-binding proteins had a preference for maltodextrins larger than maltohexaose and a low affinity for maltose and maltotriose. Both the degradative enzymes and starch binding were induced by maltose. These findings indicate that starch utilization by B. thetaiotaomicron apparently does not involve secretion of extracellular enzymes. Rather, binding of the starch molecule to the cell surface appears to be a first step to passing the molecule through the outer membrane and into the periplasmic space.

Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron

“…4A), supports the hypothesis that the transposon insertion in Ms-1 interrupts a regulatory locus that controls expression of many starch utilization genes. Ms-S had also appeared to be a regulatory mutant because of its aberrant expression of starch-degrading enzymes (2). If the Tn4351 insertion in Ms-S has in fact disrupted a regulatory locus, this locus does not control expression of the four maltose-inducible proteins detected on the Western blots.…”

“…An unusual feature of the B. thetaiotaomicron starch utilization system is that the degradative enzymes are not extracellular but are located in the periplasm or cytoplasm (2,14). Some membrane-bound activity was detected, but the enzymes appeared not to be exposed on the cell surface.…”

“…Moreover, binding activity appeared to be regulated similarly to that of the degradative enzymes. That is, high-level binding activity was seen in bacteria grown on maltose but not in bacteria grown on glucose or other monosaccharides (2).…”

“…First, binding of labelled starch to the surface of B. thetaiotaomicron was demonstrated and was found to be protease sensitive, saturable, and specific for long stretches of a-1,4-linked glucose residues (2). Thus, it appeared that there might be a starch-specific receptor complex on the surface of the bacteria.…”

Bacteroides: the Good, the Bad, and the Nitty-Gritty

“…At sites of infection, B. fragilis may utilize host cell surface glycoproteins and glycolipids as a nutrient source; these may include simple sugars such as galactose and mannose and more complex compounds (e.g., N-acetyl-D-glucosamine [NAG]) and N-acetylneuraminic acids). Indeed, the largest paralogous group of proteins in B. thetaiotaomicron are those involved in oligo-and polysaccharide uptake and degradation (2,3,58,59,72,73,156,206,207,229,247,248,276), capsular biosynthesis, and environmental sensing/signal transduction/DNA mobilization (306). The authors of the B. thetaiotaomicron genome sequence publication suggest that these expansions "reveal strategies used by B. thetaiotaomicron to survive and to dominate in the densely populated intestinal system" (306).…”