Journal: Cell

Article Title: Interspecies competition impacts targeted manipulation of human gut bacteria by fiber-derived glycans

doi: 10.1016/j.cell.2019.08.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: On the day of gavage, stocks of these strains were thawed in an anaerobic chamber and mixed together along with each of five multi-taxon INSeq libraries ( B. thetaiotaomicron VPI-5482, B. thetaiotaomicron 7330, B. cellulosilyticus WH2, B. vulgatus ATCC-8482, B. ovatus ATCC-8483) whose generation and characterization have been described in earlier publications ( Hibberd et al., 2017 ; Wu et al., 2015 ).

Techniques: Recombinant, BIA-KA, Gel Extraction, dsDNA Assay, Software, Molecular Weight

Journal: bioRxiv

Article Title: Microbiota functional activity biosensors for characterizing nutrient utilization in vivo

doi: 10.1101/2020.11.02.362798

Figure Lengend Snippet: Isolation of an arabinose-rich fraction from pea fiber . (A) Mole fraction of neutral monosaccharides from PFABN and SBABN. Mean values ± s.d. from triplicate measurements are shown. (B) Proposed structure of arabinan isolated from pea fiber and sugar beet as determined by glycosyl linkage analysis. The colored arabinose monosaccharides highlight the different glycosyl branching patterns found in arabinan isolated from the two sources. (C) Growth curves of four Bacteroides strains cultured in minimal medium containing either glucose or arabinan as the sole carbon source. Data from samples where no exogenous carbon source was added are subtracted from all curves. Solid lines represent the mean and shaded area the s.e.m. of quadruplicate cultures (data shown are representative of 3 independent experiments). (See also Supplementary file 1 ).

Article Snippet: Five of the Bacteroides ( B. thetaiotaomicron strains VPI-5482 and 7330, B. vulgatus ATCC 8482, B. cellulosilyticus WH2, B. ovatus ATCC 8483) were each represented by previously described libraries of tens of thousands of transposon (Tn) insertion mutants ( ; ).

Techniques: Isolation, Cell Culture

Journal: bioRxiv

Article Title: Microbiota functional activity biosensors for characterizing nutrient utilization in vivo

doi: 10.1101/2020.11.02.362798

Figure Lengend Snippet: Assessing the biological activity of PFABN in gnotobiotic mice colonized with a defined consortium of human gut bacterial strains . (A) Experimental design. Germ-free mice were fed the unsupplemented HiSF-LoFV diet for 5 days then colonized with the indicated group of 14 bacterial strains (the five Bacteroides strains represented in the form of Tn mutant libraries are highlighted in boldface). Two days after gavage of the consortium different groups of animals were switched to a HiSF-LoFV diet supplemented with raw pea fiber, PFABN or SBABN and fed that diet monotonously for 10 days (average dose of supplement consumed/per day is shown). Control animals were maintained on the unsupplemented HiSF-LoFV diet. (B) Absolute abundances of supplement-responsive bacterial strains, plus the total bacterial load of all 14 strains in fecal samples obtained at the indicated time points (each dot represents a single animal; bar height represents the mean; error bars represent s.d.). *, p<0.01 [generalized linear mixed-effects model (Gaussian); two-way ANOVA with Tukey’s HSD, FDR-corrected]. (C) Specific activity of each diet supplement on the summed total absolute abundances of the four diet-responsive Bacteroides . Open circles represent mean values and error bars the s.e.m. of two independent experiments (n=10-11 mice/treatment arm). *, p<0.01 for comparisons defined by the horizontal lines [generalized linear mixed-effects model (Gaussian); two-way ANOVA with Tukey’s HSD, FDR-corrected]. (See also Supplementary files 2-4 ). The online version of this article includes the following figure supplement(s) for Figure 2: Figure supplement 1. The effects of supplementing the HiSF-LoFV diet with unfractionated pea fiber, PFABN or SBABN on PUL gene expression. Figure supplement 2. Identifying PULs that function as key fitness determinants in the different diet contexts.

Article Snippet: Five of the Bacteroides ( B. thetaiotaomicron strains VPI-5482 and 7330, B. vulgatus ATCC 8482, B. cellulosilyticus WH2, B. ovatus ATCC 8483) were each represented by previously described libraries of tens of thousands of transposon (Tn) insertion mutants ( ; ).

Techniques: Activity Assay, Mutagenesis, Control, Gene Expression

Journal: bioRxiv

Article Title: Microbiota functional activity biosensors for characterizing nutrient utilization in vivo

doi: 10.1101/2020.11.02.362798

Figure Lengend Snippet: Identifying PULs that function as key fitness determinants in the different diet contexts . Plots represent of the log 2 fitness score versus log 2 fold-change in protein abundance for all genes from a given organism, ( A ) B. thetaiotaomicron VPI-5482, ( B ) B. vulgatus ATCC 8482, ( C ) B. ovatus ATCC 8483 and ( D ) B. cellulosilyticus WH2, under the specified diet condition. Genes from the specified PUL are highlighted in blue. The overrepresentation of genes positioned in the right lower quadrants of the plots, (i.e., those showing high expression and low fitness when they are disrupted by a transposon), was defined with a chi-squared test using all other genes with both proteomic and INSeq data as the null. The central shaded region represents an ellipse of the inter-quartile range of both the fitness score and protein abundance for that organism under the specified diet condition. This region was excluded from the chi-squared calculation of a PUL being overrepresented in in the lower right quadrant to increase the stringency of the test. Presented P-values are FDR-corrected.

Article Snippet: Five of the Bacteroides ( B. thetaiotaomicron strains VPI-5482 and 7330, B. vulgatus ATCC 8482, B. cellulosilyticus WH2, B. ovatus ATCC 8483) were each represented by previously described libraries of tens of thousands of transposon (Tn) insertion mutants ( ; ).

Techniques: Quantitative Proteomics, Expressing

Journal: bioRxiv

Article Title: Microbiota functional activity biosensors for characterizing nutrient utilization in vivo

doi: 10.1101/2020.11.02.362798

Figure Lengend Snippet: Degradation of MFAB-bound PFABN by B. thetaiotaomicron VPI-5482 and B. cellulosyliticus WH2 in vitro . ( A,B ) Input beads (panel A) were incubated with B. thetaiotaomicron VPI-5482 that had been grown in BMM medium to mid-log phase with glucose as the carbon source. Cells were harvested and resuspended in BMM with or without different concentrations of supplemented destarched PFABN. PFABN-MFABs were added and the mixture was incubated at 37 °C: aliquots of the reaction mixture were withdrawn at the time points shown (panel B). ( C-E ) Input beads (panel C) were incubated with B. cellulosilyticus WH2 that had been grown to mid-log phase in BMM containing glucose (panel D) or PFABN (panel E) as the carbon sources. Cells were harvested and resuspended in BMM with or without different concentrations of destarched PFABN. MFABs were harvested at the indicated time points and the percentage of arabinose remaining on the bead was calculated from the mean absolute mass of arabinose on the input preparation of MFABs used in that experiment. Each point in panels A-E represents a single measurement (n=6 or 8 for input beads; 3 or 4 for beads exposed to the Bacteroides strains). Mean values ± s.d. are plotted. The green ‘X’ denotes the mean number (triplicate determinations) of colony forming units (CFU) in the incubation mixture at time of bead harvest (see right y-axis).

Article Snippet: Five of the Bacteroides ( B. thetaiotaomicron strains VPI-5482 and 7330, B. vulgatus ATCC 8482, B. cellulosilyticus WH2, B. ovatus ATCC 8483) were each represented by previously described libraries of tens of thousands of transposon (Tn) insertion mutants ( ; ).

Techniques: In Vitro, Incubation

Journal: bioRxiv

Article Title: Microbiota functional activity biosensors for characterizing nutrient utilization in vivo

doi: 10.1101/2020.11.02.362798

Figure Lengend Snippet: Quantifying microbial degradation of PFABN- and SBABN-coated beads in gnotobiotic mice fed unsupplemented or supplemented HiSF-LoFV diets. (A) Monosaccharide composition of beads containing covalently bound PFABN or SBABN. Control beads were subjected to surface amine acetylation. The amount of monosaccharide released after acid hydrolysis was quantified by GC-MS. Each point represents a single measurement. Bar height denotes the mean while error bars represent the s.d. (n=6 measurements). (B,C) Percentage of arabinose, galactose and xylose remaining on the surface of beads recovered from the cecums of mice fed the indicated diets (n=5 mice/treatment group). *, p<0.05 (Mann-Whitney U test compared to the group furthest to the left). (See also Supplementary file 5 ). The online version of this article includes the following figure supplement(s) for Figure 4: Figure supplement 1. Enzyme degradation of PFABN immobilized on an MFAB surface using CDAP chemistry. Figure supplement 2. Degradation of MFAB-bound PFABN by B. thetaiotaomicron VPI-5482 and B. cellulosyliticus WH2 in vitro . Figure supplement 3. Assaying whether bead-linked polysaccharides are degraded in germ-free mice.

Article Snippet: Five of the Bacteroides ( B. thetaiotaomicron strains VPI-5482 and 7330, B. vulgatus ATCC 8482, B. cellulosilyticus WH2, B. ovatus ATCC 8483) were each represented by previously described libraries of tens of thousands of transposon (Tn) insertion mutants ( ; ).

Techniques: Control, Gas Chromatography-Mass Spectrometry, MANN-WHITNEY, In Vitro

Journal: bioRxiv

Article Title: Microbiota functional activity biosensors for characterizing nutrient utilization in vivo

doi: 10.1101/2020.11.02.362798

Figure Lengend Snippet: Colocalization of PFABN and glucomannan on the same bead results in augmented degradation of glucomannan in gnotobiotic mice colonized with the defined consortium and fed the pea fiber supplemented HiSF-LoFV diet. (A) In vitro growth of supplement-responsive Bacteroides species in minimal medium containing glucose or glucomannan as the sole carbon source. Data from samples where no exogenous carbon source was added are subtracted from all curves. The line represents the mean and shaded regions the s.e.m. of quadruplicate measurements. (B) Monosaccharide compositions of beads with covalently bound PFABN, glucomannan, or both PFABN and glucomannan. Control beads were subjected to surface amine acetylation. The amount of monosaccharide released after acid hydrolysis was quantified by GC-MS. Each point represents a single measurement. Bar height represents the mean and error bars the s.d. (n=6 measurements). (C) Beads containing PFABN alone, glucomannan alone, or both glycans, as well as ‘empty’ acetylated control beads, each containing a unique fluorophore, were simultaneously introduced by oral gavage into gnotobiotic mice, recovered 4 hours later from their cecums. Each bead-type is subsequently purified by FACS. A representative flow cytometry plot of beads isolated from the cecum is shown. (D) Monosaccharide remaining on beads coated with PFABN alone, glucomannan alone, or both glycans after collection and purification from the cecums of mice fed the unsupplemented or pea fiber-supplemented HiSF-LoFV diet. Colors are identical to those used in panel b. The amount of remaining monosaccharide is expressed relative to the absolute mass of monosaccharide immobilized on the surface of each type of input bead. Each point represents a single animal. *, p<0.05 (Mann-Whitney U test). (See also Supplementary file 5 ).

Article Snippet: Five of the Bacteroides ( B. thetaiotaomicron strains VPI-5482 and 7330, B. vulgatus ATCC 8482, B. cellulosilyticus WH2, B. ovatus ATCC 8483) were each represented by previously described libraries of tens of thousands of transposon (Tn) insertion mutants ( ; ).

Techniques: In Vitro, Control, Gas Chromatography-Mass Spectrometry, Purification, Flow Cytometry, Isolation, MANN-WHITNEY