Polysaccharide utilization loci in Bacteroides determine population fitness and community-level interactions

Abstract

Polysaccharide utilization loci (PULs) are co-regulated bacterial genes that sense nutrients and enable glycan digestion. Human gut microbiome members, notably Bacteroides, contain numerous PULs that enable glycan utilization and shape ecological dynamics. To investigate the role of PULs on fitness and inter-species interactions, we develop a CRISPR-based genome editing tool to study 23 PULs in Bacteroides uniformis (BU). BU PULs show distinct glycan-degrading functions and transcriptional coordination that enables the population to adapt upon loss of other PULs. Exploiting a BU mutant barcoding strategy, we demonstrate that in vitro fitness and BU colonization in the murine gut are enhanced by deletion of specific PULs and modulated by glycan availability. PULs mediate glycan-dependent interactions with butyrate producers that depend on the degradation mechanism and glycan utilization ability of the butyrate producer. Thus, PULs determine community dynamics and butyrate production and provide a selective advantage or disadvantage depending on the nutritional landscape.

Keywords: Bacteroides; CRISPR-based genome editing; butyrate production; microbial interactions; microbial metabolism; microbiome engineering; polysaccharide utilization loci; synthetic human gut communities; transcriptional regulation.

Figure 1.. Effects of polysaccharide utilization loci (PULs) on growth of B. uniformis in response to individual glycans.

(A) Schematic of the two-stage procedure to construct the barcoded PUL deletion mutants using the CRISPR-FnCpf1 genome editing tool. First, the barcode-tagged mutants were constructed and then each PUL was deleted using FnCpf1. Gray square brackets indicates that the plasmid used to delete a subset of PULs harbored recT. The growth of each mutant was characterized in media with individual glycans. Plasmid features include p15A, E. coli plasmid replication origin; pB8–51, Bacteroides plasmid replication origin; lacI, transcription factor; P_lacO23, IPTG inducible promoter. (B) Biclustering heatmap of the fold changes in steady-state abundance (k_i in the logistic growth model) for each PUL mutant to ΔtryP-24 in media with single carbon sources. Asterisks represent low confidence (coefficient of variation > 0.2) in the inferred parameter value (Supplementary Data 2). In these conditions, the steady-state abundance was set to the maximum OD₆₀₀. (C) Bipartite network of PULs and glycans based on thresholds in the fold changes of inferred steady-state abundance and/or growth rate of each PUL deletion mutant to ΔtryP-24. (D) Time-series measurements of OD₆₀₀ of PUL mutants in media with specific glycans highlighted in the bipartite network in (C). Lines denote the mean and the shaded regions represent 95% confidence interval of 3 biological replicates.

Figure 2.. Co-regulation of polysaccharide utilization loci (PULs) for xyloglucan utilization in B. uniformis.

(A) Schematic of gene organization of PUL11, PUL43 and PUL44. GenBank locus tags are shown for each gene. SusC: SusC-like TonB-dependent transporter (Red); SusD: SusD-like cell-surface glycan-binding protein (Pink); SBP, sugar binding protein (Purple); HTCS, hybrid two-component system (Blue). All genes encoding glycoside hydrolyses are shaded in Green. Genes with unknown functions are shaded in gray. (B) Time-series measurements of OD₆₀₀ of PUL mutants and ΔtryP-24 in media with glucose (top) or xyloglucan (bottom). Lines denote the mean and shaded regions represent 95% confidence interval of 3 biological replicates. (C) Bar plots of reads per kilobase per million mapped reads (RPKM) for each gene in PUL11, PUL43 and PUL44 in ΔtryP-24 in media with glucose or xyloglucan. Data points denote 2 biological replicates. The colored bars represent mean RPKM value of the genes shown in the same order as panel (A). (D) Bar plot of the log2 fold changes of RPKM of ΔPUL11 to ΔtryP-24 (left), ΔPUL43 to ΔtryP-24 (middle), or ΔPUL11_44 to ΔtryP-24 (right) in xyloglucan media (n=2, *p<0.05, unpaired t-test). (E) Bar plot of the fold change of BACUNI_RS15340 in PUL43 to ΔtryP-24 in xyloglucan media using qRT-PCR (n = 6, ***p<0.001; ΔPUL11 vs ΔtryP-24 p=2.9e-5; ΔPUL11_44 vs ΔtryP-24 p=2.3e-5, unpaired t-test). Bars indicate the mean of replicates +/− 1 s.d. and data points show individual measurements. (F) Heatmap of log2 fold changes of RPKM for PUL genes in ΔPUL11, ΔPUL43, ΔPUL44 or ΔPUL11_44 compared to ΔtryP-24 in xyloglucan media. The x-axis represents individual genes contained in the indicated PULs. The indicated PULs (x-axis) contained at least one gene with an absolute value of the log2 fold change greater than 2.

Figure 3.. Impact of polysaccharide utilization loci (PULs) on B. uniformis pooled mutant fitness in media with different carbon sources.

(A) Schematic representing experimental design of the pooled barcoded PUL mutants and ΔtryP-24 in media with different carbon sources. The mutant pool was passaged every 24 hr using a 1:20 dilution. The absolute abundance of each mutant was determined by barcode sequencing and OD₆₀₀ measurements. (B) Stacked bar plots represent the mean relative abundance of each mutant in media with a given carbon source (top). Bar plots denote the OD₆₀₀ of each condition (bottom). Data points represent 3 biological replicates. (C) Violin plots represent the distribution of the absolute abundance of the PUL mutants in media with a given carbon source. Colored data points represent the absolute abundance of the indicated mutant within this distribution (n = 3 biological replicates). (D) Heatmap of Shannon diversity of mutant pool across different passages. The values represent the mean of 3 biological replicates.

Figure 4.. Impact of polysaccharide utilization loci (PULs) on the colonization ability of B. uniformis in germ-free mice fed different diets.

(A) Schematic of experimental design to evaluate the effect of PULs on BU colonization ability in germ-free mice fed different diets. Top: mice were fed a high fiber diet, fiber free diet (FFD) or high fat diet a week prior to oral gavage and then maintained on the same diet for two weeks following oral gavage (n = 5 for high fiber group and n = 4 for other groups). Bottom: mice were fed the FFD a week prior to oral gavage and then provided with drinking water supplemented with inulin, pectic galactan or glucomannan (n = 4). On day 0, mice were orally gavaged with the mutant pool and ΔtryP-24. Time-series measurements of fecal samples were performed. The cecal samples were collected at the end of the experiment. (B) Stacked bar plot of the absolute abundance of mutants in cecal samples (CFU g⁻¹) in each group of mice. Data points denote 2 independent CFU measurements for each mouse. The two most abundance mutants are highlighted. Asterisks denote a statistically significant difference in the CFU of each group compared to FFD group based on unpaired t-test (n = 8; *p<0.05, ***p<0.001; High fiber diet vs FFD, p=4.9e-9; High fat diet vs FFD, p=0.01271; FFD-Inulin vs FFD, p=2.8e-6; FFD-Glucomannan vs FFD, p=1.0e-4; FFD-Pectic galactan vs FFD, p=0.01393). (C) (i) Stacked bar plots of relative abundance of mutants in fecal samples as a function of time (top). Line plots of Shannon diversity of the mutant pool as a function of time (bottom). Asterisks indicate statistically significant difference of Shannon diversity of each group on day 14 compared to FFD group based on unpaired t-test (n=4–5; **p<0.01, ***p<0.001; High fiber diet vs FFD, p=3.9e-6; High fat diet vs FFD, p=1.4e-4; FFD-Glucomannan vs FFD, p=0.00425; FFD-Pectic galactan vs FFD, p=7.9e-5). (ii) Categorial scatter plot of the maximum slope of the Shannon diversity as a function of time. The colored data points represent individual mice and the black data point represents the mean. (D) Principal component analysis (PCA) as a function of time. Colors represent different diets based on the legend in (A). The size of the data points is proportional to the time of measurement. The PCA loadings are denoted by the black lines. Symbols represent different mice. (E) Line plots of the relative abundance of PUL mutants or ΔtryP-24 in mice fed different diets as a function of time. The colors represent different diets based on the legend in (A). Data points denote individual mice and lines represent the mean. The asterisks denote a statistically significant difference in relative abundance between the FFD and each indicated diet based on unpaired t-test (n=4–5, p<0.05).

Figure 5.. B. uniformis polysaccharide utilization loci (PULs) mediate glycan-dependent inter-species interactions influencing butyrate production.

(A) Schematic representing the experimental design investigating PUL mediated inter-species interactions between Inter-species interactions were deduced using a generalized Lotka-Volterra (gLV) model informed by time-series data of species absolute abundance. Representative data (middle) shows time-series measurements of absolute abundance based on 16S rDNA sequencing and OD₆₀₀ measurements in inulin media (Fig. S7). Species include BU WT (blue), BU PUL mutant (purple) and butyrate producers (BPs): A. caccae (AC, orange), C. comes (CC, green), E. rectale (ER, yellow) and R. intestinalis (RI, red). The color of each growth curve indicates the species. The first row represents monoculture conditions and the second and third rows denote the growth of each species in the BU WT, butyrate producer (BP) or BU PUL mutant, BP coculture in inulin media. (B) Inferred networks of inter-species interactions between BU WT or a given PUL mutant and each BP in media with different carbon sources. Node size represents the maximum mean OD₆₀₀ measured in monoculture in the indicated media. For species whose maximum mean OD₆₀₀ was less than 0.5, their node sizes were set to OD₆₀₀=0.5 for visibility in the network. The width of an edge connecting node j to node i represents the magnitude of the median of the inferred marginal distribution of their inter-species interaction coefficient (a_ij). An edge is colored red (blue) if the interaction is positive (negative). If the 25% and the 75% quantiles of the a_ij marginal distribution have different signs, we represent the edge with a dashed line, indicating lack of certainty. Inter-species interactions where the magnitude of the median of the marginal a_ij distribution is less than 0.01 are not included in the network. (C) Scatter plot of predicted and measured butyrate concentrations in monoculture and coculture experiments. Predicted butyrate concentrations are computed according to the linear regression model (Methods). Marker horizontal position represents predicted butyrate concentration based on mean end-point BP abundance, and horizontal error bars represent 1 s.d. of predicted butyrate concentration given the uncertainty in BP abundance measurements.

Figure 6.. Mechanisms of PUL mediated interactions between B. uniformis (BU) and butyrate producers.

(A) Schematic of Mechanism A: (i) butyrate producer (BP) such as A. caccae (AC) is unable to utilize the given glycan but can utilize PBPs released by BU. (ii) Deletion of a given PUL such as PUL17 eliminates release of PBPs and thus AC is unable to grow in co-culture with ΔPUL17 in inulin media. Bar plot of absolute abundance (B) and butyrate production (C) of BU WT, ΔPUL17 and A. caccae (AC) monoculture and coculture in inulin media after 48 hr of growth. (D) Fructose concentrations in inulin or BU conditioned inulin media. (E) Growth of AC in glucose and fructose media. Lines denote the mean and the shaded regions represent 95% confidence interval. (F) Schematic of Mechanism B: (i) BP such as R. intestinalis (RI) can utilize pullulan and thus compete with BU. (ii) RI can grow in co-culture with ΔPUL18 in pullulan media. Bar plot of absolute abundance (G) and butyrate production (H) of BU WT, ΔPUL18 and RI monoculture and coculture in pullulan media after 48 hr of growth. (I) Schematic of Mechanism C: (i) BP such as C. comes (CC) lacks the capability to utilize both the given glycan and PBPs. (ii) CC is unable to grow in coculture with ΔPUL11_43 in xyloglucan media. Bar plot of absolute abundance (J) and butyrate production (K) of BU WT, ΔPUL11_43 and CC monoculture and co-culture in xyloglucan media after 48 hr of growth. (L) Bar plot of the fold change of the area under curve (AUC) of CC in conditioned xyloglucan media and cell membrane treated xyloglucan media to AUC of CC in xyloglucan media. Bars indicate the mean of replicates ± 1 s.d. and data points show individual measurements (n>=3 biological replicates). PBPs: Polysaccharide breakdown products.