The WildR murine gut microbiome is stable over multiple generations and harbors an ICE-encoded T6SS

(A) Phylum-level taxonomic composition of the WildR or lab-derived murine gut microbiome at different generations. Data from the initial wild-caught donor mice (Wild), the WildR F2 generation and the lab mice-derived community derive from new analysis of previously published sequencing data (Rosshart et al. 2017). WildR F7 data derives from two cryopreserved samples of pooled ilocecal contents (A, B) and fecal pellets from two mice (1, 2) used to propagate the community. *indicates technical duplicate samples. Source data is presented in Supplemental Table 1. (B-E) Comparison of the abundance of the 100 genera most prevalent in wild donor mice between the indicated communities. Points are shaded to show overlapping datapoints (darker shades), and Spearman’s correlation for each comparison is indicated (ρ). ND, not detected. (F) To-scale schematic depicting the Bacteroides acidifaciens T6SS gene cluster. (G) Bioinformatic domain predictions for T6SS effector and immunity proteins of B. acidifaciens. Conserved amino acids are indicated in bold.

The B. acidifaciens T6SS intoxicates other WildR Bacteroidales species.

(A) Normalized growth yield (final/initial CFU, mean ± SD) of the indicated strains of B. acidifaciens (recipients) after in vitro growth in competition with B. acidifaciens wild-type or ΔtssC (donors). Data are representative of at least three biological replicates. *p ≤ 0.05 (two-tailed t-test). CFU, colony forming units; bae, e; bai, i. (B) Mean (± SD) relative fitness of wild-type B. acidifaciens relative to a ΔtssC derivative during in vitro growth competition assays with the indicated species isolated from the WildR community. Data represent 3 biological replicates. *indicates the T6SS is significantly important for competitiveness (unpaired two-tailed t-test. p < 0.05). (C) Abundance (% reads per kilobase per million) of WildR species in two initial wild-caught donor mice used to establish the WildR (Rosshart et al. 2017). (D) Abundance of B. acidifaciens Δbae1 Δbai1 Δbae2 Δbai2 in mouse fecal samples collected following co-gavage of germ-free mice with this strain and wild-type or T6SS-inactivated (ΔtssC) B. acidifaciens. ND, not detected. *p < 0.05 (mixed-model ANOVA with repeated measures and Šidák’s multiple comparisons tests) (E) Relative abundance (compared to total Bacteroides) of B. caecimuris F5 following co-gavage of germ-free mice with B. acidifaciens wild-type or ΔtssC. *p < 0.05 (two-way ANOVA with repeated measures and Šidák’s multiple comparisons tests). For D and E, n=6 mice from two independent replicates. Boxplots represent the interquartile range and mean for each condition; whiskers represent minimum and maximum detectable values; points represent individual values.

Maintenance of B. acidifaciens in the WildR community is mediated by the T6SS

(A) Schematic of approach to exploit the carrying capacity of the mouse gut to promote modified strain engraftment during WildR microbiome establishment without affecting community structure. Left, design of proof-of-concept experiment to evaluate the effect of increasing the amount of B. acidifaciens relative to the amount of the WildR microbiome during oral gavage. Right, design of experiment to evaluate the importance of the T6SS for B. acidifaciens fitness in the WildR microbiome-colonized mouse gut. (B-D) Analysis of total (B), or introduced (C,D) B. acidifaciens populations in gavage (Day 0) or post-gavage fecal samples from germ-free mice colonized with the WildR and variable levels of B. acid exo. B) Total abundance of B. acidifaciens calculated from sequencing 16S rRNA genes amplified from DNA extracted from fecal samples. Differences in B. acidifaciens abundance across mice gavaged with different amounts of B. acid exo was not significant (n=4 mice/sample, mixed-effects analysis). C) Relative abundance of B. acid exo in the indicated fecal samples compared to total B. acidifaciens as determined by qPCR. D) Abundance of B. acid exo in the indicated fecal samples as determined by plating for CFU on selective media. (E) Principal coordinate analysis of weighted Unifrac diversity metrics calculated from 16S rRNA gene amplicon sequencing data from feces collected from mice colonized with the WildR and variable amounts of B. acid exo. (F-H) Quantification of B. acid exo (F,G) or total (H) B. acidifaciens population in gavage (Day 0) and post-gavage fecal samples from germ-free mice colonized with the WildR community and 10-fold excess wild-type or ΔtssC B. acid exo. n=8 mice per strain, across two biological replicates. (F) CFU quantification of B. acid exo and B. acid exo ΔtssC. *p ≤ 0.05 (two-way ANOVA with repeated measures and Šidák’s multiple comparisons test). (G) Abundance of B. acid exo or B. acid exo ΔtssC relative to the total B. acidifaciens population, as determined by qPCR. *p ≤ 0.05 (two-way ANOVA with repeated measures and Šidák’s multiple comparisons test). (H) Total abundance of B. acidifaciens, calculated from 16S rRNA gene amplicon sequencing. Samples from mice colonized by the WildR community alone (no B. acid exo, white bars) are included for comparison. Differences in B. acidifaciens abundance based on B. acid exo genotype were not significant (mixed-model ANOVA test). For panels B-D and F-H, boxplots represent the interquartile range with indicated mean for each condition, whiskers represent maximum and minimum detectable values, and points show values from individual mice.

P. vulgatus gains a limited fitness benefit from the T6SS-encoding ICE in WildR-colonized mice.

(A) Frequency of mapped ICE junctions deriving from the indicated species as determined by 5′ or 3′ ICE-Seq analysis of DNA extracted from fecal samples collected either 7 or 14 days post-gavage of the WildR into germ-free mice (n=4). Mice housed in separate or shared cages are indicated. (B) Relative competitive index from in vitro growth competitions between P. vulgatus ICE or ICE ΔtssC and recipient strains isolated from the WildR microbiome (Pseudomonadata, grey; Bacteroidota, green). The mean ± SD from three biological replicates is shown. Asterisks indicate recipient species for which the difference in competitive index was statistically significant between donor strains (p<0.05, unpaired two-tailed t-test). (C) Recovery of P. vulgatus strains containing the indicated versions of the ICE after in vitro growth with B. acidifaciens. The mean ± SD from one biological replicate and its associated technical replicates are shown, which represent results from at least three biological replicates. *p ≤ 0.05 (two-tailed t-test). bae, e; bai, i. (D) Abundance of wild-type P. vulgatus in feces collected from mice (n=4) co-colonized with P. vulgatus ICE or P. vulgatus ICE ΔtssC. Boxplots represent the interquartile range with indicated mean for each condition, whiskers represent minimum and maximum values, points show values from individual mice. *p ≤ 0.05 (two-way ANOVA with repeated measures test and Šidák’s multiple comparisons test). (E) Schematic of experimental design to test the fitness of P. vul exo + T6SS-ICE in the WildR community. (F) Recovery of P. vul exo + ICE (dark blue) or P. vul exo + ICE ΔtssC (light blue) from post-gavage fecal samples of mice co-colonized with the WildR community as depicted in (E). Boxplots represent the interquartile range with indicated mean for each condition, whiskers represent minimum and maximum values, points show values from individual mice. Data in panel F is from two biological replicates with 4 mice per group per replicate (n=8). *p < 0.05 (two-way ANOVA with repeated measures and Šidák’s multiple comparisons test to compare P.vulgatus ICE and ICE ΔtssC populations), † p < 0.05 (unpaired t-test to compare day 7 and day 56 samples).

Evidence supporting stability of the WildR microbiome over multiple generations and identification of an ICE-encoded T6SS

(A) Schematic depicting the generation of the WildR F7 cryoprserved stocks from the combined cecal contents of six mice used to propagate the community, and subsequent community characterization steps. (B-C) Comparison of the abundance of the 100 genera most prevalent in wild donor mice between the indicated communities. Points are shaded to show overlapping datapoints (darker shades), and Spearman’s correlation for each comparison is indicated (ρ). ND, not detected. (D) Mapping efficiency of metagenomic reads from different WildR community generations to selected murine-derived genome databases: the WildR catalog (86 MAGs and genomes from the WildR, generated in this study) or the comprehensive mouse microbiota genome catalog (CMMG; 1573 species across mouse microbiomes (Kieser et al. 2022)). (E) To-scale schematic of the ICE containing the GA1 T6SS encoded in B. acidifaciens and B. caecimuris F12. Location of single base deletion in B. caecimuris F12 highlighted in the orange box.

Activity of the B. acidifaciens T6SS against species co-resident in the WildR community.

(A) In vitro mating efficiency of the integrative plasmid pNBU2-ermG:: tssC into B. acidifaciens. “RM silent” indicates plasmid was mutated to remove a B. acidifaciens methylated motif. Data shown are mean ± SD from 3 independent matings. N.D., not detected; D.L., detection limit. (B) Recipient abundance after in vitro growth competitions between B. acidifaciens donors lacking various structural components of the T6SS and the indicated recipient species. (C) Competitive index from in vitro growth competition between indicated WildR isolates (recipient) and B. acidifaciens donors. For B & C, data show the mean ± SD of technical replicates from one biological replicate and represent results from at least three biological replicates. *p ≤ 0.05 by two-tailed t-test; all other comparisons were not significant. (D) B. caecimuris F5 abundance in cecal contents from germ-free mice co-colonized with B. acidifaciens wild-type or ΔtssC. Data show the mean ± SD and points indicate values from individual mice (n=6) across two biological replicates. *p ≤ 0.05 (two-tailed t-test). (E) P. vulguatus abundance in feces from germ-free mice (n=6, two biological replicates) co-colonized with B. acidifaciens wild-type or ΔtssC and P. vulguatus. Boxplots represent the interquartile range with indicated mean for each condition, whiskers represent minimum and maximum values, points represent individual values. *p ≤ 0.05 (two-way ANOVA with repeated measures test and Šidák’s multiple comparisons test). (F) P. vulguatus abundance in cecal contents from mice described in panel E. Data show the mean ± SD and points show values from individual mice (n=6). No statistical difference was found based on B. acidifaciens genotype by two-tailed t-test. (G) Relative abundance (% reads per kb per million) of selected WildR Bacteroidales species in the WildR F7 generation. Data are mean + SD from cryopreserved WildR stocks and 2 fecal samples from mice used to propagate the WildR F7 community.

Addition of B. acid exo to the WildR does not alter community composition and enables in situ measurement of T6SS-mediated fitness.

(A,B) Recovery of B. acid exo from feces (A) and cecal contents (B) following gavage of germ-free mice with the WildR community and the indicated amount of B. acid exo relative to the endogenous population. (C,D) Recovery of B. acid exo or B. acid exo ΔtssC from feces (D) or cecal contents (E) from mice colonized with the WildR and B. acid exo strains. *p ≤ 0.05 (two-way ANOVA with repeated measures test and Šidák’s multiple comparisons test in C, unpaired t-test in D). For data in panels A-D, boxplots represent the interquartile range with indicated mean for each condition, whiskers represent minimum and maximum values, and points show values from individual mice. (E) Principal coordinate analysis of weighted Unifrac diversity metrics calculated from 16S rRNA amplicon sequencing data from feces collected from mice colonized with the WildR alone or in combination with the indicated strain of B. acid exo. Gavage samples highlighted in pink and remaining timed fecal and cecal (collected at 56 days post gavage) samples are colored as indicated. Data shown are from one biological replicate, representative two experiments conducted.

Distribution of the T6SS-ICE in the WildR suggests limited fitness benefit to some Bacteroides sp.

(A) Schematic of ICE-seq approach to identify WildR species encoding the ICE. The junction amplification and sequencing strategy applied to both ends of the ICE is depicted only for the 3′ end for simplicity. (B) Schematic depicting ICE transfer from B. acidifaciens (marked with CmR) to P. vulgatus (marked with ErmR) via in vitro conjugation and selective plating. (C) To-scale schematic of ICE insertion sites in P. vulgatus exo + ICE transconjugants that acquired the indicated versions of the ICE. (D) Abundance (relative to total P. vulgatus) of the indicated P. vulgatus exo in fecal samples from mice co-colonized with the WildR community, as determined by qPCR. Boxplots represent the interquartile range with indicated mean for each condition, whiskers represent minimum and maximum values, and points show values from individual mice (n=8) from two biological replicates. Asterisks indicate significant differences between P. vul exo + ICE and P. vul exo + ICE ΔtssC frequency at the indicated time points (p<0.05, Šídák’s multiple comparisons test, mixed model ANOVA with multiple comparisons). N.D., not detected. (E) Principal coordinate analysis of weighted Unifrac diversity metrics calculated from 16S rRNA gene amplicon sequencing data from feces collected from mice (n=8/group across 2 biological replicates) colonized with the WildR and either P. vul exo + ICE (closed circles) or P. vul exo + ICE ΔtssC (open circles). The community composition varied between groups at early time points (purple; Rep 1, p=0.021, pseudo-F=2.7; Rep 2, p=0.011, pseudo-F=12, PERMANOVA test), but varied less or not signifcantly at late time points (orange; Rep 1, p=0.25, pseudo-F=1.3; Rep 2, p=0.003, pseudo-F=5).