An Ecological Network of Polysaccharide Utilization among Human Intestinal Symbionts  Seth Rakoff-Nahoum, Michael J. Coyne, Laurie E. Comstock  Current Biology  Volume 24, Issue 1, Pages 40-49 (January 2014) DOI: 10.1016/j.cub.2013.10.077 Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 1 Variation among Bacteroidales Members in Ability to Utilize Polysaccharides and to Publicly Liberate PBPs (A) Growth curves of bacteria in defined media with indicated polysaccharide as carbohydrate source. (B) Thin-layer chromatography (TLC) analyses of polysaccharide breakdown products in the CM of primary utilizers through the growth phases (1, early; 2, mid; 3, late log; 4, stationary) in defined media with indicated polysaccharide as carbohydrate source. The intact polysaccharides do not migrate from the bottom of the TLC plate. PBPs are continually generated as they are consumed during bacterial growth due to continued breakdown of polysaccharide. Fru, fructose; FOS, fructose oligosaccharides (derived from inulin); Glu, glucose; Malt, maltose; MD, maltodextrin. Data are representative of at least three independent experiments; one representative experiment is shown. See also Figure S1. Current Biology 2014 24, 40-49DOI: (10.1016/j.cub.2013.10.077) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 2 Specific Non-Polysaccharide-Utilizing Bacteroidales Members Can Utilize Liberated PBP Public Goods (A) Growth curves of non-primary-utilizing bacteria in media conditioned by PBP-liberating utilizers. (B) TLC analysis of culture supernatants of recipient bacteria grown in the CM of primary utilizers. As P. distasonis did not grow in Bo-inulin CM, its time points correspond to the growth points of B. vulgatus in the same CM through the growth phases (1, mid; 2, late log; 3, stationary). (C) Growth curves of B. vulgatus in inulin media conditioned by different species of inulin-utilizing Bacteroidales. (D) TLC analysis of culture supernatants of B. vulgatus grown in B. caccae and B. uniformis inulin CM through the growth phases (1, mid; 2, late log; 3, stationary). Data are representative of at least three independent experiments; one representative experiment is shown. See also Figure S2. Current Biology 2014 24, 40-49DOI: (10.1016/j.cub.2013.10.077) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 3 Growth and PBP Liberation by Recipients Containing Producer-Derived GH/PL Genes (A) Growth curves of recipient bacteria with genes encoding the B. ovatus inulin PLs (BACOVA_04502 and BACOVA_04503), the B. thetaiotaomicron levan GH (BT_1760), the B. thetaiotaomicron amylopectin GH (susG, BT_3698), or vector alone (pFD340) in defined polysaccharide media. The initial decrease in OD600 in amylopectin and levan media correspond to rapid degradation of these optically dense polysaccharides. (B) TLC analyses of PBPs released from recipient strains containing GH/PL genes in trans or vector alone (pFD340) through the growth phases (Inulin and amylopectin: 1, early; 2, mid; 3, late log; 4, stationary. Levan: 1, lag; 2, early; 3, mid; 4, late log; 5, stationary). As recipient with vector alone did not grow in polysaccharide media, its time points correspond to the growth points of recipient strains containing GH/PL genes in trans. Data are representative of at least independent experiments; one representative experiment is shown. Current Biology 2014 24, 40-49DOI: (10.1016/j.cub.2013.10.077) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 4 GH/PLs Serve as Public Goods through Secretion in Outer Membrane Vesicles (A) Left: growth of utilizers on defined amylopectin agarose plates demonstrating amylopectin degraded zones surrounding Bf, Bo, and Bt, demonstrating extracellular release of GH. Middle: growth capabilities of recipient (Bv) and late recipient (Bc) plated at various distances (a, b, c, and d) from the producer B. ovatus on a defined amylopectin plate. B. vulgatus is only able to grow in the zone of amylopectin degradation, whereas late recipient, B. caccae does not (left). Right: growth of recipient B. vulgatus (spotted on all 36 spots except 3C) is dictated by its spatial proximity to the producer B. ovatus (spotted on 3C) on a defined inulin plate. (B) TLC analyses of GH/PL activity in culture supernatants of PBP-liberating utilizer strains grown in indicated defined medium with extra polysaccharide added and incubated over time. The polysaccharide at the origin of the TLC is degraded with accumulation of PBPs. (C) TLC analysis of extracellular GH/PL activity from recipient strains with GH/PL genes in trans or vector alone. Bacteria were cultured in defined glucose medium without polysaccharide as the GH/PL genes are expressed from a constitutive plasmid-borne promoter. Supernatants were harvested, filter sterilized, and diluted 1:1 with medium containing the indicated polysaccharide and incubated at 37°C over time prior to TLC analysis. The glucose at the top of the TLC is from the initial growth medium. Glu, glucose. (D) Growth of B. vulgatus but not P. distasonis in defined inulin medium with purified BACOVA_04502 and BACOVA_04503 added to the medium. B. vulgatus does not grow with material purified from the vector only control. (E) TLC analysis of the resulting media from the samples shown in (D), through the growth phases (1, lag; 2, lag; 3, late log; 4, stationary), demonstrating PBP consumption by B. vulgatus. “C” indicates B. vulgatus grown with material prepared from vector-only control. As this B. vulgatus with vector control material and P. distasonis did not grow in Bo-inulin CM, its time points correspond to the growth points of B. vulgatus with inulinases through the growth phases. (F) Western immunoblot analysis of cell lysates (CL), supernatant (sup), or outer-membrane vesicles (OMVs) from wild-type transconjugants synthesizing His-tagged GH/PLs. BT_1760 was not tracked in this assay. (G) Growth of B. vulgatus in defined inulin medium with added OMVs isolated from B. ovatus inulin CM. The OMVs were harvested from supernatant of B. ovatus grown to log phase so that the bacteria were actively growing at the time of harvest. (H) TLC analysis of the resulting media from the samples shown in (G), through the growth phases (1, early; 2, mid; 3, late log), demonstrating PBP consumption by B. vulgatus during growth in OMV + inulin media. The first lane is B. vulgatus cultured inulin medium without OMV at the same time point as B. vulgatus with OMV at time point 3. Data are representative of at least two independent experiments; one representative experiment is shown. See also Figure S3. Current Biology 2014 24, 40-49DOI: (10.1016/j.cub.2013.10.077) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 5 Ecological Classes and Network of Polysaccharide Utilization (A) Schematic diagram designating Bacteroidales type strains to one of five ecological classes: utilizer/public good producer, utilizer/public good nonproducer, PBP public good recipient (inducible), PBP public good recipient (noninducible), and nonrecipient. (B) A network of interactions based on PS utilization for Bacteroidales type strains. See also Figure S4. Current Biology 2014 24, 40-49DOI: (10.1016/j.cub.2013.10.077) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 6 Fitness Assays of Polysaccharide-Utilizing and -Nonutilizing Strains in Coculture Growth of type strains in coculture or monoculture in defined inulin medium (A) or defined amylopectin medium (B). The B ovatus strain is an arginine auxotrophic mutant, allowing for analyses under both growth limiting (low arginine, 1 μg/ml) and nonlimiting (high arginine, 16 μg/ml) conditions. Solid lines represent bacterial counts from monoculture experiments, whereas dotted lines represent bacterial counts from coculture experiments. Monoculture experiments for recipients were performed under low-arginine conditions only, as their growth is not affected by arginine; therefore, this growth curve is used for both producer-limited and not limited experiments. The limit of detection (indicated with ∗ for strains that were below detection) for a given experiment is set at 2 logs below the total density of the culture. Data are representative of at least three independent experiments; one representative experiment is shown. Comparison of growth rates was as follows. Inulin producer nonlimited: Bv in monoculture (−0.08 ± 1.17) versus Bv in coculture (4.62 ± 0.05), p = 0.027. Bo in monoculture (5.58 ± 0.24) versus Bo in coculture with Bv (7.24 ± 2.4), p = 0.30, not significant (ns). Relative frequency producer: recipient (93.9% ± 2.4%:6.1% ± 2.4%). Inulin producer limited: Bv in monoculture (−0.08 ± 1.17) versus Bv in coculture (3.9 ± 1.2), p = 0.005. Relative frequency producer: recipient (5.3% ± 2.1%:94.7% ± 2.1%). Bo in monoculture (3.14 ± 0.37) versus Bo in coculture with Bv (3.08 ± 1.1), p = 0.48, ns. Amylopectin producer nonlimited: Bv in monoculture (−2.67 ± 0.73) versus Bv in coculture (4.69 ± 2.54), p = 0.02. Bo in monoculture (6.06 ± 1.13) versus Bo in coculture with Bv (9.34 ± 0.89), p = 0.02. Relative frequency producer: recipient (96% ± 1%:4% ± 1%). Pd in monoculture (−1.95 ± 1.07) versus Pd in coculture (1.42 ± 1.56), p = 0.05. Bo in monoculture (6.07 ± 1.13) versus Bo in coculture with Pd (9.88 ± 0.52), p = 0.045. Relative frequency producer: recipient (92% ± 2.7%:8% ± 2.7%). Amylopectin producer limited: Bv in monoculture (−2.7 ± 1.15) versus Bv in coculture (11.2 ± 4.1), p = 0.02. Bo in monoculture (6.4 ± 1.77) versus Bo in coculture with Bv (6.22 ± 1.08), p = 0.42, ns. Relative frequency producer: recipient (4.6% ± 2.7%:95.4% ± 2.7%). Pd in monoculture (−1.82 ± 1.23) versus Pd in coculture (5.84 ± 3.35), p = 0.87, ns. Bo in monoculture (6.37 ± 1.78) versus Bo in coculture with Pd (4.9 ± 0.23), p = 0.21. Relative frequency producer: recipient (15.7% ± 7.5%:84.4% ± 7.5%). Limits of detection precluded determination of growth rate of Pd in inulin coculture and Bc in amylopectin coculture. See also Figure S5. Current Biology 2014 24, 40-49DOI: (10.1016/j.cub.2013.10.077) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 7 Polysaccharide-Based Ecological Relationships of Naturally Coresident Bacteroidales (A) Growth curves of naturally coresident Bacteroidales strains from human subject CL03 in the four defined polysaccharide media. (B) TLC analysis of PBP release during growth of CL03 strains in defined polysaccharide media. (C and D) Growth (C) and TLC (D) analysis of PBP consumption by CL03-nonutilizing strains grown in the CM of primary utilizers, through the growth phases (1, early; 2, mid; 3, late log). Producer-derived CM was diluted with fresh polysaccharide containing defined media for recipient growth to assess inducible polysaccharide utilization. Therefore, lane 0 (undiluted producer CM) has less polysaccharide than the subsequent lanes. Data are representative of at least two independent experiments; one representative experiment is shown. Current Biology 2014 24, 40-49DOI: (10.1016/j.cub.2013.10.077) Copyright © 2014 Elsevier Ltd Terms and Conditions