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The Bacteroides thetaiotaomicron starch utilization system (Sus) is a model system for nutrient acquisition by gut Bacteroidetes, a dominant phylum of gut bacteria. The Sus includes SusCDEFG, which assemble on the cell surface to capture, degrade and import starch. While SusD is an essential starch-binding protein, the precise role(s) of the partially homologous starch-binding proteins SusE and SusF has remained elusive. We previously reported that a non-binding version of SusD (SusD*) supports growth on starch when other members of the multi-protein complex are present. Here we demonstrate that SusE supports SusD* growth on maltooligosaccharides, and determine the domains of SusE essential for this function. Furthermore, we demonstrate that SusE does not need to bind starch to support growth in the presence of SusD*, suggesting that the assembly of SusCDE is most important for maltooligosaccharide uptake in this context. However, starch binding by proteins SusDEF directs the uptake of maltooligosaccharides of specific lengths, suggesting that these proteins equip the cell to scavenge a range of starch fragments. These data demonstrate that the assembly of core Sus proteins SusCDE is secondary to their glycan binding roles, but glycan binding by Sus proteins may fine tune the selection of glycans from the environment.

The Bacteroides thetaiotaomicron starch utilization system (Sus) is a model system for nutrient acquisition by gut Bacteroidetes, a dominant phylum of gut bacteria. The Sus includes SusCDEFG, which assemble on the cell surface to capture, degrade and import starch. While SusD is an essential starch-binding protein, the precise role(s) of the partially homologous starch-binding proteins SusE and SusF has remained elusive. We previously reported that a non-binding version of SusD (SusD*) supports growth on starch when other members of the multi-protein complex are present. Here we demonstrate that SusE supports SusD* growth on maltooligosaccharides, and determine the domains of SusE essential for this function. Furthermore, we demonstrate that SusE does not need to bind starch to support growth in the presence of SusD*, suggesting that the assembly of SusCDE is most important for maltooligosaccharide uptake in this context. However, starch binding by proteins SusDEF directs the uptake of maltooligosaccharides of specific lengths, suggesting that these proteins equip the cell to scavenge a range of starch fragments. These data demonstrate that the assembly of core Sus proteins SusCDE is secondary to their glycan binding roles, but glycan binding by Sus proteins may fine tune the selection of glycans from the environment.

Summary Bacteroides thetaiotaomicron (Bt) is a human colonic symbiont that degrades many different complex carbohydrates (glycans), the identities and amounts of which are likely to change frequently and abruptly from meal-to-meal. To understand how this organism reacts to dynamic growth conditions, we challenged it with a series of different glycan mixtures and measured responses involved in glycan catabolism. Our results demonstrate that individual Bt cells can simultaneously respond to multiple glycans and that responses to new glycans are extremely rapid. The presence of alternative carbohydrates does not alter response kinetics, but reduces expression of some glycan utilization genes as well as the cell's sensitivity to glycans that are present in lower concentration. Growth in a mixture containing 12 different glycans revealed that Bt preferentially uses some before others. This metabolic hierarchy is not changed by prior exposure to lower priority glycans because re-introducing high priority substrates late in culture re-initiates repression of genes involved in degrading those with lower priority. At least some carbohydrate prioritization effects occur at the level of monosaccharide recognition. Our results provide insight into how a bacterial glycan generalist modifies its responses in dynamic glycan environments and provide essential knowledge to interpret related metabolic behaviour in vivo.

Summary Xenorhabdus nematophila colonizes the intestinal tract of infective-juvenile (IJ) stage Steinernema carpocapsae nematodes. During colonization, X. nematophila multiplies within the lumen of a discrete region of the IJ intestine termed the vesicle. To begin to understand bacterial nutritional requirements during multiplication in the IJ vesicle, we analysed the colonization behaviour of several X. nematophila metabolic mutants, including amino acid and vitamin auxotrophs. X. nematophila mutants defective for para-aminobenzoate, pyridoxine or l-threonine biosynthesis exhibit substantially decreased colonization of IJs (0.1–50% of wild-type colonization). Analysis of gfp-labelled variants revealed that those mutant cells that can colonize the IJ vesicle differ noticeably from wild-type X. nematophila. One aberrant colonization phenotype exhibited by the metabolic mutants tested, but not wild-type X. nematophila, is a spherical shape indicative of apparently non-viable X. nematophila cells within the vesicle. Because these spherical cells appear to have initiated colonization but failed to proliferate, we term this type of colonization ‘abortive’. In a portion of IJs grown on para-aminobenzoate auxotrophs, X. nematophila does not exhibit abortive colonization but rather reduced growth and filamentous cell morphology. Several mutants with defects in other amino acid, vitamin and nutrient metabolism pathways colonize IJs to wild-type levels suggesting that the IJ vesicle is replete with respect to a number of nutrients.

Xenorhabdus nematophila colonizes the intestinal tract of infective-juvenile (IJ) stage Steinernema carpocapsae nematodes. During colonization, X. nematophila multiplies within the lumen of a discrete region of the IJ intestine termed the vesicle. To begin to understand bacterial nutritional requirements during multiplication in the IJ vesicle, we analysed the colonization behaviour of several X. nematophila metabolic mutants, including amino acid and vitamin auxotrophs. X. nematophila mutants defective for para-aminobenzoate, pyridoxine or l-threonine biosynthesis exhibit substantially decreased colonization of IJs (0.1-50% of wild-type colonization). Analysis of gfp-labelled variants revealed that those mutant cells that can colonize the IJ vesicle differ noticeably from wild-type X. nematophila. One aberrant colonization phenotype exhibited by the metabolic mutants tested, but not wild-type X. nematophila, is a spherical shape indicative of apparently non-viable X. nematophila cells within the vesicle. Because these spherical cells appear to have initiated colonization but failed to proliferate, we term this type of colonization 'abortive'. In a portion of IJs grown on para-aminobenzoate auxotrophs, X. nematophila does not exhibit abortive colonization but rather reduced growth and filamentous cell morphology. Several mutants with defects in other amino acid, vitamin and nutrient metabolism pathways colonize IJs to wild-type levels suggesting that the IJ vesicle is replete with respect to a number of nutrients.