Research ArticleGUT MICROBIOTA

The effects of micronutrient deficiencies on bacterial species from the human gut microbiota

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Science Translational Medicine  17 May 2017:
Vol. 9, Issue 390, eaal4069
DOI: 10.1126/scitranslmed.aal4069
  • Fig. 1. The effect of dietary micronutrient deficiency on the configuration of a defined human gut microbiota established in gnotobiotic mice.

    (A) Experimental design. (B) Principal coordinates analysis of pairwise comparisons of fecal microbiota using Bray-Curtis dissimilarities of Wisconsin square root–transformed abundance data obtained from COPRO-Seq analysis. Fecal samples were obtained from mice in the indicated treatment groups at the indicated time points. Gray shaded ellipses and spokes indicate the SEM of sample group centroids from the vitamin A–deficient and the micronutrient-sufficient (monotonous diet control) groups in each experimental phase. PCo 1, principal coordinate 1. (C) COPRO-Seq analysis of the effects of the micronutrient-deficient diets versus micronutrient-sufficient diets on the abundance of B. vulgatus and Bacteroides dorei in the fecal microbiota of gnotobiotic mice. Means ± SEM. **P < 0.01, ***P < 0.001 [one-way analysis of variance (ANOVA), Tukey’s honest significant difference (HSD), FDR correction; n = 5 mice per treatment group].

  • Fig. 2. The distinct retinol sensitivity phenotypes of B. vulgatus strains and B. dorei in vitro.

    (A) Growth curves of B. vulgatus in defined medium with and without various retinoids. The horizontal dashed line indicates the threshold used for calculating time–to–log phase measurements. (B and C) Bar plots indicating mean (±SEM) retinoid sensitivity, calculated as time–to–log phase for treated cultures versus time–to–log phase for vehicle alone (DMSO) control cultures for B. vulgatus ATCC 8482 versus B. dorei DSM 17855 (B) or B. vulgatus strain ATCC 8482 versus B. vulgatus strain 257_H4 (C) isolated from a healthy Malawian infant (16). For example, the sensitivity value of 20.8 ± 2.1 for wild-type B. vulgatus incubated in medium containing 10 μM retinol in (B) was calculated by dividing the total incubation period (in this case, 95 hours) by the time required for vehicle alone–treated control cultures of the same strain to cross the OD600 threshold of 0.3. Means ± SEM are shown except under those conditions in (C), where the concentration of retinol tested completely inhibited growth. n = 2 independent experiments, each in triplicate for (B), and 1 experiment performed in triplicate for (C). **P < 0.01, ***P < 0.001 (one-way ANOVA, Bonferroni multiple comparisons test).

  • Fig. 3. Selection of retinol-resistant B. vulgatus Tn mutants.

    (A) Experimental design. The mutant library was inoculated into defined medium containing 10 μM retinol or 0.02% (v/v) DMSO (three cultures per treatment). In the first round of selection, mutant libraries were allowed to grow to stationary phase and were then passaged to fresh medium and subjected to a second round of selection. Aliquots were withdrawn in lag and stationary phases from the primary cultures and in log and stationary phases of the secondary cultures. The site of insertion of the Tn was defined in the retinol-resistant mutants using INSeq. (B) Percent abundance of Tn mutants in retinol-selected B. vulgatus libraries. The left portion of the panel indicates the abundance of each selected mutant in the input library. Each set of four bars shown in the right portion of the panel indicates the abundance of the Tn mutants at the indicated growth phases from both primary and passaged cultures. (C) Schematic of the B. vulgatus locus containing the retinol-resistant mutants identified from screening the Tn library. Annotation is based on the National Center for Biotechnology Information reference assembly NC_009614.1. The genomic location of each selected Tn mutant is indicated by a downward pointing arrow annotated with the corresponding color from (B) and the corresponding genome coordinate for the site of Tn insertion. (D) Schematic of components comprising the E. coli AcrAB-TolC efflux system (adapted from http://2013.igem.org/Team:Ciencias-UNAM/Project). (E) Retinol sensitivity of B. vulgatus wild-type (WT) and Tn mutants grown in monoculture in defined medium treated with 1, 5, and 10 μM retinol versus 0.02% (v/v) DMSO as a reference vehicle control. Means ± SEM of the ratio between treated and control cultures for each strain are shown. The sensitive B. vulgatus WT strain and resistant B. dorei WT strain are shown as positive and negative controls, respectively. (F) Retinol (10 μM) sensitivity of the WT, acrR::IN (genome location 361472) mutant, and complemented B. vulgatus acrR::IN + pNBU2_acrR mutant strains (abbreviated Comp), plus a control B. vulgatus acrR::IN strain containing the empty vector. The results shown in (E) are from two independent experiments, each performed in triplicate, whereas those in (F) are from three independent experiments, each performed in triplicate or quadruplicate. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA, Bonferroni multiple comparisons test).

  • Fig. 4. Transcriptional response of the B. vulgatus locus containing the AcrAB-TolC efflux pump to insertional mutagenesis of acrR or lpxA.

    Microbial RNA-seq analysis of gene expression in mid-log phase B. vulgatus WT, acrR::IN, and lpxA::IN (genome location 362422) strains cultured in the absence of retinol. Transcript counts, normalized by DESeq2, for each gene in the putative BVU0244-BVU0233 operon are shown. Bars indicate means ± SEM for n = 3 independent cultures of each strain. Significant differences in gene expression were defined by DESeq2 analysis. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 5. Interactions between the AcrR transcription factor and its target DNA binding site.

    (A) Predicted AcrR-regulated operons in the genomes of human gut Bacteroides species. Boxes indicate clusters of coregulated genes. Filled black circles indicate predicted AcrR-binding sites. Orthologous gene symbols are indicated for each species; unnamed genes are indicated using the orthologous B. vulgatus locus designation. (B) Sequences of predicted AcrR-binding sites. BVU, B. vulgatus; BACDOR, B. dorei; BT, B. thetaiotaomicron. (C) Consensus binding site motif. (D) Electrophoretic mobility shift assay of the interactions between AcrRBV and AcrRBD and their predicted target DNA sequences.

  • Fig. 6. Role of the AcrAB-TolC efflux pump in regulating the sensitivity of B. vulgatus to retinol and deoxycholic acid.

    (A) Retinol efflux assay. Stationary phase cultures of the WT, acrR::IN, and complemented acrR::IN + pNBU2_acrR strains were resuspended in PBS containing cysteine and 10 μM retinol. Samples were collected over a 2-hour time period, and retinol concentrations in cell-free supernatants were quantified by UPLC-MS. Four independent experiments were performed; n = 1 to 3 replicates per experiment (two-way, repeated-measures ANOVA, Tukey’s HSD test). (B) Sensitivity of the WT, acrR::IN, and acrR-complemented strains of B. vulgatus and WT B. dorei to 10 μM retinol in the presence and absence of PAβN, a chemical inhibitor of multidrug efflux systems (see table S15A for further details). Data represent one experiment, performed in triplicate (one-way ANOVA, Bonferroni multiple comparisons test). (C) Sensitivity of B. vulgatus and B. dorei strains to deoxycholic acid (DCA) in the presence and absence of PAβN. Data represent one experiment, performed in triplicate (one-way ANOVA, Bonferroni multiple comparisons test). Means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/390/eaal4069/DC1

    Fig. S1. Comparisons of community structure across experimental stages.

    Fig. S2. Relative abundances of B. vulgatus ATCC 8482 and B. dorei DSM 17855 in gnotobiotic mice across experimental stages and treatment groups.

    Fig. S3. Characterization of the B. vulgatus ATCC 8482 INSeq library.

    Fig. S4. Maximum likelihood phylogenetic tree of BVU0240/AcrR orthologs identified in human gut–associated Bacteroides and other members of the family Bacteroidaceae.

    Fig. S5. DNA binding characteristics of AcrRBV and AcrRBD in the presence and absence of possible effectors.

    Table S1. Nutritional characteristics of experimental micronutrient-deficient and micronutrient-sufficient diets.

    Table S2. Ninety-two sequenced, human gut–derived bacterial strains.

    Table S3. COPRO-Seq analysis of community composition in fecal samples.

    Table S4. Influence of micronutrient deficiencies on the relative abundances of specific taxa.

    Table S5. Identification of community members that exhibit significant changes in their abundance as a function of diet treatment and/or time.

    Table S6. Microbial RNA-seq analysis of changes in community metatranscriptome as a function of diet treatment with grouping of transcripts into KO groups.

    Table S7. Microbial RNA-seq analysis of changes in B. vulgatus gene expression as a function of diet treatment with grouping of transcripts into KO groups.

    Table S8. Strain-level microbial RNA-seq analysis of the effects of vitamin A on gene expression (summarized at the level of KO groups).

    Table S9. Mouse weights as a function of diet treatment and time.

    Table S10. In vitro retinoid sensitivities of Bacteroides strains.

    Table S11. Strains, primers, and plasmids used in this study.

    Table S12. Microbial RNA-seq analysis of differential gene expression between wild-type B. vulgatus and Tn mutants (DESeq2).

    Table S13. Bioinformatic characterization of AcrR regulons in human gut Bacteroides strains.

    Table S14. High-resolution quantitative MS-based proteomic analysis of wild-type B. vulgatus cultured in the presence of 1 μM retinol versus vehicle alone (0.02% DMSO).

    Table S15. Effects of retinol, bile acids, and PAβN on growth of wild-type and mutant strains of B. vulgatus and wild-type B. dorei.

    Table S16. UPLC-MS analysis of the effects of dietary micronutrient deficiency on fecal bile acid metabolites.

  • Supplementary Material for:

    The effects of micronutrient deficiencies on bacterial species from the human gut microbiota

    Matthew C. Hibberd, Meng Wu, Dmitry A. Rodionov, Xiaoqing Li, Jiye Cheng, Nicholas W. Griffin, Michael J. Barratt, Richard J. Giannone, Robert L. Hettich, Andrei L. Osterman, Jeffrey I. Gordon*

    *Corresponding author. Email: jgordon{at}wustl.edu

    Published 17 May 2017, Sci. Transl. Med. 9, eaal4069 (2017)
    DOI: 10.1126/scitranslmed.aal4069

    This PDF file includes:

    • Fig. S1. Comparisons of community structure across experimental stages.
    • Fig. S2. Relative abundances of B. vulgatus ATCC 8482 and B. dorei DSM 17855 in gnotobiotic mice across experimental stages and treatment groups.
    • Fig. S3. Characterization of the B. vulgatus ATCC 8482 INSeq library.
    • Fig. S4. Maximum likelihood phylogenetic tree of BVU0240/AcrR orthologs identified in human gut–associated Bacteroides and other members of the family Bacteroidaceae.
    • Fig. S5. DNA binding characteristics of AcrRBV and AcrRBD in the presence and absence of possible effectors.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Nutritional characteristics of experimental micronutrient-deficient and micronutrient-sufficient diets.
    • Table S2 (Microsoft Excel format). Ninety-two sequenced, human gut–derived bacterial strains.
    • Table S3 (Microsoft Excel format). COPRO-Seq analysis of community composition in fecal samples.
    • Table S4 (Microsoft Excel format). Influence of micronutrient deficiencies on the relative abundances of specific taxa.
    • Table S5 (Microsoft Excel format). Identification of community members that exhibit significant changes in their abundance as a function of diet treatment and/or time.
    • Table S6 (Microsoft Excel format). Microbial RNA-seq analysis of changes in community metatranscriptome as a function of diet treatment with grouping of transcripts into KO groups.
    • Table S7 (Microsoft Excel format). Microbial RNA-seq analysis of changes in B. vulgatus gene expression as a function of diet treatment with grouping of transcripts into KO groups.
    • Table S8 (Microsoft Excel format). Strain-level microbial RNA-seq analysis of the effects of vitamin A on gene expression (summarized at the level of KO groups).
    • Table S9 (Microsoft Excel format). Mouse weights as a function of diet treatment and time.
    • Table S10 (Microsoft Excel format). In vitro retinoid sensitivities of Bacteroides strains.
    • Table S11 (Microsoft Excel format). Strains, primers, and plasmids used in this study.
    • Table S12 (Microsoft Excel format). Microbial RNA-seq analysis of differential gene expression between wild-type B. vulgatus and Tn mutants (DESeq2).
    • Table S13 (Microsoft Excel format). Bioinformatic characterization of AcrR regulons in human gut Bacteroides strains.
    • Table S14 (Microsoft Excel format). High-resolution quantitative MS-based proteomic analysis of wild-type B. vulgatus cultured in the presence of 1 μM retinol versus vehicle alone (0.02% DMSO).
    • Table S15 (Microsoft Excel format). Effects of retinol, bile acids, and PAβN on growth of wild-type and mutant strains of B. vulgatus and wild-type B. dorei.
    • Table S16 (Microsoft Excel format). UPLC-MS analysis of the effects of dietary micronutrient deficiency on fecal bile acid metabolites.

    [Download Tables S1 to S16]