Ecological Importance of Cross-Feeding of the Intermediate Metabolite 1,2-Propanediol between Bacterial Gut Symbionts

Abstract

Cross-feeding based on the metabolite 1,2-propanediol has been proposed to have an important role in the establishment of trophic interactions among gut symbionts, but its ecological importance has not been empirically established. Here, we show that in vitro growth of Lactobacillus reuteri (syn. Limosilactobacillus reuteri) ATCC PTA 6475 is enhanced through 1,2-propanediol produced by Bifidobacterium breve UCC2003 and Escherichia coli MG1655 from the metabolization of fucose and rhamnose, respectively. Work with isogenic mutants showed that the trophic interaction is dependent on the pduCDE operon in L. reuteri, which encodes the ability to use 1,2-propanediol, and the l-fucose permease (fucP) gene in B. breve, which is required for 1,2-propanediol formation from fucose. Experiments in gnotobiotic mice revealed that, although the pduCDE operon bestows a fitness burden on L. reuteri ATCC PTA 6475 in the mouse digestive tract, the ecological performance of the strain was enhanced in the presence of B. breve UCC2003 and the mucus-degrading species Bifidobacterium bifidum The use of the respective pduCDE and fucP mutants of L. reuteri and B. breve in the mouse experiments indicated that the trophic interaction was specifically based on 1,2-propanediol. Overall, our work established the ecological importance of cross-feeding relationships based on 1,2-propanediol for the fitness of a bacterial symbiont in the vertebrate gut.IMPORTANCE Through experiments in gnotobiotic mice that employed isogenic mutants of bacterial strains that produce (Bifidobacterium breve) and utilize (Lactobacillus reuteri) 1,2-propanediol, this study provides mechanistic insight into the ecological ramifications of a trophic interaction between gut symbionts. The findings improve our understanding on how cross-feeding influences the competitive fitness of L. reuteri in the vertebrate gut and revealed a putative selective force that shaped the evolution of the species. The findings are relevant since they provide a basis to design rational microbial-based strategies to modulate gut ecosystems, which could employ mixtures of bacterial strains that establish trophic interactions or a personalized approach based on the ability of a resident microbiota to provide resources for the incoming microbe.

Keywords: 1,2-propanediol; Lactobacillus; bifidobacteria; competition; cross-feeding; fitness; gut microbiome; metabolism; microbial ecology; trophic interactions.

FIG 1

Impact of 1,2-propanediol on growth and metabolism of L. reuteri PTA 6475 and L. reuteri ΔpduCDE. (A) L. reuteri strains were grown in half-strength mMRS supplemented with either glucose (Glc; 25 mM), 1,2-propanediol (50 mM), or a mixture of the two. Asterisks indicate a significant difference (two-way ANOVA; P < 0.001) in the growth of L. reuteri PTA 6475 on glucose plus 1,2-propanediol compared to the other conditions. (B and C) Utilization of 1,2-propanediol and production of propanol and propionate by L. reuteri PTA 6475 (B) and L. reuteri ΔpduCDE (C) during growth on glucose in the presence of 1,2-propanediol. (D and E) Production of acetate (D) and ethanol (E) by the two strains during growth on glucose in the presence of 1,2-propanediol.

FIG 2

Growth and metabolites of L. reuteri PTA 6475 and L. reuteri ΔpduCDE in conditioned media of B. breve UCC2003 and B. breve UCC2003-fucP grown with cellobiose alone or with the addition of fucose. (A and B) Growth curves of L. reuteri strains in B. breve UCC2003 conditioned media (A) and L. reuteri in B. breve UCC2003-fucP conditioned media (B). Asterisks indicate a significant difference (two-way ANOVA; P < 0.001) in growth of L. reuteri PTA 6475 grown in B. breve UCC2003 conditioned medium that had fermented cellobiose and fucose together compared to growth of L. reuteri PTA 6475 in B. breve UCC2003 conditioned medium without fucose or to L. reuteri ΔpduCDE grown in all conditioned media derived from B. breve UCC2003. (C and D) Utilization of B. breve derived 1,2-propanediol present in the conditioned media and production of propanol in cultures of L. reuteri PTA 6475 (C) and L. reuteri ΔpduCDE (D) grown in the conditioned medium of B. breve UCC2003 grown in the presence of fucose. Propionate, acetate, and ethanol concentrations could not be determined due to interference of unknown compounds in the medium. Abbreviations: BM, B. breve UCC2003 conditioned media; B-fucP M, B. breve UCC2003-fucP conditioned media; (C), preculture fermentations of cellobiose only; (CF), preculture fermentations of cellobiose with added fucose (see Table 1 for more details about the media used in this study).

FIG 3

Growth curves and metabolites of L. reuteri PTA 6475 and L. reuteri ΔpduCDE in conditioned media of E. coli grown with glucose or rhamnose. (A) Growth of L. reuteri strains. Asterisks indicate a significant difference (two-way ANOVA; P < 0.01) in growth of L. reuteri PTA 6475 grown in conditioned medium from E. coli grown on rhamnose compared to conditioned medium from E. coli grown on glucose as well as growth of L. reuteri ΔpduCDE grown in conditioned medium from E. coli grown with either glucose or rhamnose. (B and C) Utilization of E. coli-derived 1,2-propanediol and production of propanol and propionate by L. reuteri PTA 6475 (B) and L. reuteri ΔpduCDE (C) grown in conditioned medium from E. coli grown with rhamnose. (D) Comparison of acetate production by the two strains grown in conditioned medium from E. coli grown with rhamnose. Ethanol concentrations could not be determined due to interference of an unknown compound in the medium. Abbreviations: EM, E. coli conditioned media; (G), preculture fermentation of glucose only by E. coli; (R), preculture fermentation of rhamnose only by E. coli (see Table 1 for more details about the media used in the study).

FIG 4

Graphical illustration of hypothesized trophic interactions of 1,2-propanediol in gnotobiotic mice. (A) In triple-species associated gnotobiotic mice (colonized by B. bifidum, B. breve, and L. reuteri), B. bifidum liberates fucose from the degradation of host mucin, which is metabolized by B. breve UCC2003 producing 1,2-propanediol, that is subsequently utilized by L. reuteri PTA 6475. (B) In dual-species (E. coli and L. reuteri) associated mice whose diet has been supplemented with rhamnose added through the drinking water, E. coli metabolizes rhamnose, producing 1,2-propanediol that is subsequently utilized by L. reuteri PTA 6475.

FIG 5

Populations of L. reuteri PTA 6475 and L. reuteri ΔpduCDE in the gastrointestinal tract of triple-species and double-species associated gnotobiotic mice. (A) Normalized ratios between L. reuteri PTA 6475 to L. reuteri ΔpduCDE obtained from Bifidobacterium-L. reuteri triple-species associated gnotobiotic mice in which colonization by L. reuteri PTA 6475 and L. reuteri ΔpduCDE was tested separately. A “+” indicates mice colonized with B. breve UCC2003, and a “–” indicates mice colonized with B. breve UCC2003-fucP. (B) Percent CFU for L. reuteri PTA 6475 and L. reuteri ΔpduCDE as measured in triple-species associated gnotobiotic mice in which the two L. reuteri strains were tested in competition. (C) Normalized ratios between L. reuteri PTA 6475 and L. reuteri ΔpduCDE in E. coli-L. reuteri double-species associated gnotobiotic mice in which colonization of L. reuteri PTA 6475 and L. reuteri ΔpduCDE was tested separately. (D) Percent CFU for L. reuteri PTA 6475 and L. reuteri ΔpduCDE mutant in double-species associated gnotobiotic mice in which the two L. reuteri strains were tested in competition. A “+” indicates the presence of rhamnose (Rha) in the diet, while a “–” indicates absence of rhamnose in the diet. Statistical significance for ratios and percent abundance (% CFU) was determined using a Mann-Whitney test and a Fisher exact test, respectively.