Dynamic responses of Bacteroides thetaiotaomicron during growth on glycan mixtures

Abstract

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.

Figure 1

A. Scatter plots of fluorescent labeling intensities of cells exposed to various combinations of glucose, AP and/or PG for two hours. A dashed red line indicates the threshold used to exclude background levels for each antibody, which was set at the high end of the MM-glucose distributions to exclude >97.5% of the glucose-grown cells and nearly all of the cells in the no antibody control. Numbers in each quadrant represent percent of the total population (n= 600 individual cells; 200 from each of three replicates). Axes are labeled with the fluorescent antibody used and units are in normalized fluorescence intensity values (arbitrary units, a.u.). See Fig. S2 for histogram representations of the same data. B. The average fluorescence intensities of Bt populations grown in the various conditions from A. Shown are mean ± standard error. Here the average background intensities for unlabeled cells were subtracted from each condition because they were disproportionately higher (2.8-fold) in the AP staining condition. Letters over each bar indicate data groupings in which the t test p-values were < 0.001. There was slight but significant activation of the AP system in the PG only condition, suggesting that this substrate contains a small amount of starch contamination. A separate qPCR assay of susC^AP expression in the ΔCPS strain under the same PG only growth conditions supports this notion by revealing that the starch system was activated 3.9±1.2 fold (p<0.05). C. Montage of fluorescently labeled Bt cells taken from the treatment conditions. All images were captured at the same exposure time (2 sec), processed identically to reduce background noise (Experimental Procedures) and are at the same scale (bar in lower right = 2 µm). In very rare cases, cells with unexpectedly high labeling intensities were observed in a condition that was not predicted to promote expression of the target protein (asterisk in second row of images). The cause of these events was not investigated further. Additional negative controls in which ΔCPS strains lacking either the susD^AP or susD^PG genes were exposed to a 1:1 mixture of AP:PG and to the individual glycans were conducted to verify that each SusD-specific antibody was specific for the product against which it was raised. In both cases, no significant fluorescence above unlabeled background was detected at the exposure times noted above (data not shown).

Figure 2

A. Activation of genes required for AP and PG metabolism is not repressed by glucose. Bt was grown to mid-exponential phase in MM-glucose (5 mg ml⁻¹), washed in 2× MM, and resuspended in either MM-AP (1 or 5 mg ml⁻¹) or MM-glucose (5 mg ml⁻¹) +AP (1 or 5 mg ml⁻¹). Transcripts were measured by qPCR 30 min after exposure for susC^AP and susC^PG and are shown relative to a MM-glucose reference. B-C. Identical experiments as described for panel A., with the remaining glycans contained in PSM, except that here only a high concentration of each glycan (5mg ml⁻¹) was tested. Graphs are separated based on whether they did not show glucose repression (B.) or did show glucose repression (C.). In all panels, values represent the mean ± standard deviation of three replicates (* p ≤ 0.001, t test).

Figure 3

Response kinetics of PUL transcripts and surface proteins when an inducing glycan is present either alone or in the context of continuous exposure to a mixture of other glycans. All induction values represent fold-change relative to a MM-glucose reference taken either at t=0 or prior to pre-growth inoculation of MM-glucose+PSM. A. susC^AP transcript expression in response to AP. B. susC^PG transcript expression in response to PG. C. SusD^AP outer membrane protein expression in response to AP. D. SusD^PG outer membrane protein expression in response to PG. In both C. and D. flow-cytometry was used to measure protein expression intensity. Symbols: response to AP or PG alone (●) or in the presence of 0.5 (□) or 5 mg ml⁻¹ (Δ) PSM. As a negative control for non-specific antibody-labeling, Bt Δ susD^AP (C.) or Δ susD^PG (D.) were grown in the presence of 5 mg ml⁻¹ PSM plus 5 mg ml⁻¹ of AP or PG, respectively (▲). The lack of detectable responses indicates that the high basal response after pre-growth on PSM is due to background contamination in the glycan mixture, rather than cross-reactivity with other SusD-like proteins that are induced by PSM. Values represent the mean ± standard deviation of three replicates.

Figure 4

Bt PUL expression is dependent on the concentration of the inducing glycan and presence of additional glycans. A. susC^AP transcript levels in Bt cells exposed for 30 min to various AP maize concentrations either alone (○) or with 5 mg ml⁻¹ PG (●), relative to MM-glucose. Transcript levels for susC^PG were also monitored when PG was present at constant 5 mg ml⁻¹ (▲). B. susC^PG transcript levels in Bt cells exposed for 30 min to various PG potato concentrations either alone (Δ) or with 5 mg ml⁻¹ AP (▲). Transcript levels for susC^AP in the AP PUL were also monitored when AP was present at 5 mg ml⁻¹ (●). Values represent the mean ± standard deviation of three replicates. Filled arrows indicate points at which susC^AP expression is reduced by increasing PG concentration.

Figure 5

Expression of Bt PULs in response to media containing AP maize with glucose, either alone or in combination with two different concentrations of PSM glycans. An additional comparison shows responses to AP maize and the higher concentration of PSM, but without glucose. Transcript levels for sentinel susC-like genes contained in PULs that target AP, PG and 9 other glycans (locus tag numbers indicated) were monitored in Bt cells that were exposed to 5 mg ml⁻¹ AP for 30 min following previous incubation in either MM-glucose, or MM-glucose containing 0.5 or 5 mg ml⁻¹ of a polysaccharide mix (PSM) lacking AP. The polysaccharide mix contained equal concentrations of dextran, chondroitin sulfate (CS), PG, heparin, homogalacturonan (HG), α-mannan (α-Mann), levan, arabinan, arabinogalactan (AG), and rhamnogalacturonan I (RGI). Values represent the mean ± standard deviation of three replicates. Letters above histogram bars are used to label measurements within each substrate category that were significantly different from one another (p < 0.05, t test).

Figure 6

Temporal expression of Bt PULs during unperturbed growth in a mixture of 12 polysaccharides (solid lines). The PSM-12 mixture contained equal amounts (0.1 mg ml⁻¹ each) of the glycans listed in Fig. 5, plus O-linked glycans harvested from porcine gastric mucin, at a final combined concentration of 1.2 mg ml⁻¹ total polysaccharides. At 6 hr post PSM-exposure, the culture was split, and one half was anaerobically washed in 2× MM then resuspended in fresh MM+PSM-12 (dashed line). Samples were removed at 30 min time points for RNA preparation. Transcript levels were determined using qPCR assays against sentinel susC-like genes representing PULs that are induced by each of the 12 polysaccharides (locus tag numbers for each gene are indicated). All transcript level changes are relative to time 0, prior to PSM-12 exposure. Two additional replicates of this time course are shown in Figs. S5–S6, two additional PSM spike-in experiments are shown in Figs. S7–S8. The 12 plots are organized in approximate order from high to low priority based on depletion of the target glycan with respect to time and the response to fresh PSM exposure. Only one of several PULs implicated in O-linked glycan metabolism is shown here (BT1280), but similar responses for three additional PULs implicated in degradation of these glycans is shown in Fig. S9.