Metabolic cross-feeding structures the assembly of polysaccharide degrading communities

Abstract

Metabolic processes that fuel the growth of heterotrophic microbial communities are initiated by specialized biopolymer degraders that decompose complex forms of organic matter. It is unclear, however, to what extent degraders structure the downstream assembly of the community that follows polymer breakdown. Investigating a model marine microbial community that degrades chitin, we show that chitinases secreted by different degraders produce oligomers of specific chain lengths that not only select for specialized consumers but also influence the metabolites secreted by these consumers into a shared resource pool. Each species participating in the breakdown cascade exhibits unique hierarchical preferences for substrates, which underlies the sequential colonization of metabolically distinct groups as resource availability changes over time. By identifying the metabolic underpinnings of microbial community assembly, we reveal a hierarchical cross-feeding structure that allows biopolymer degraders to shape the dynamics of community assembly.

Fig. 1.. Successional dynamics of chitin-degrading seawater communities.

(A) Species used in this study. Heatmap depicting (left to right) final optical density at 600 nm (OD₆₀₀) after growth on colloidal chitin (2 g/liter), final OD₆₀₀ after growth on 10 mM chitobiose, growth rate on 20 mM GlcNAc, classified functional guild, and copy numbers of genes relevant for chitin degradation. Growth data were obtained from three independent biological replicates. (B) Schematic of functional guilds: Degraders can grow on chitin as a sole carbon source through breakdown of chitin using chitin-degrading enzymes, exploiters can grow on monomeric or oligomeric N-acetylglucosamine (GlcNAc) as a sole carbon source, and scavengers require metabolites secreted by other species to be sustained in the community. (C) 16S sequences of the 18 investigated species are mapped to 16S ribosomal gene ESVs whose abundances were previously determined on chitin particles colonized from a species pool in seawater (26). Mean normalized frequencies of species that comprise each functional guild are plotted over time. TBDR, TolB-dependent receptor.

Fig. 2.. Chitin degradation products influence exploiter population and their secreted resources.

(A) Quantification of GlcNAc oligonucleotide during in vitro digestion of chitin (5 g/liter) by secreted enzymes from each degrader. (B) OD₆₀₀ of three exploiters after 36-hour growth on 20 mM GlcNAc, 10 mM chitobiose, and three enzyme digests. (C) Growth of scavengers and exploiters after 36 hours on the cell-free supernatant produced by each of the five degraders upon growth on colloidal chitin. (D) Absolute abundance of species in cocultures of one degrader (Vib1A01 or Psy6C06) and four exploiters during grown on colloidal chitin, as determined by quantitative polymerase chain reaction (qPCR). (E) Enzymatic quantification of ammonia and acetate in degrader supernatants upon growth on colloidal chitin. (F) Principal components analysis (PCA) exploiter metabolite secretion profiles after growth on each colloidal chitin digest. Colors represent the digest used as a growth substrate. All error bars represent SD from three biological replicates.

Fig. 3.. Metabolite consumption and production profiles among community members.

(A) Growth of scavengers at 36 hours on cell-free supernatants obtained from degraders and exploiters after growth on GlcNAc. (B) Metabolite concentration in cultures of degraders or exploiters after growth on 20 mM GlcNAc. α-KG, α-ketoglutarate. (C) Ammonia concentration in cultures of degraders or exploiters after growth on GlcNAc. (D) Consumption of crossfed metabolites in the pooled supernatant. (Left) FIA-QTOF-MS measurements of metabolites that are consumed by degraders, exploiters, or scavengers (ion intensity log₂ < −0.5). (Right) Time course measurements of metabolite consumption. Points represent the fraction of the final OD₆₀₀ at which metabolite consumption is detected for each species. (E) Relative change of acetate, pyruvate, GlcNAc, and glutamate during growth in the pooled supernatant by three exploiters. Curves are fit using local polynomial regression. (F) Fraction of available metabolites contained in chitin or GlcNAc-derived resource pools that is consumed by each functional guild. All experiments were performed in triplicate. Error bars represent SD. DEHEM, 4-deoxy-α-l-erythro-hex-4-enopyranuronate-β-d-mannuronate; OHCU, 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline. Strain names in green are degraders, blue are exploiters, and orange are scavengers.

Fig. 4.. Outcomes of chitin-degrading cocultures and hypothetical metabolic exchange networks.

(A) Each coculture was seeded with six species: one degrader (green), one exploiter (blue), and each of the four scavengers ParaC2R09 (yellow), CitC3M06 (orange), MarD2M19 (red), and MarF3R11 (purple). Cultures were grown for 5 weeks with four serial dilutions on colloidal chitin, and relative abundance is shown for the final culture. Experiments were performed in triplicate. (B) Two of 12 hypothetical metabolite exchange networks that illustrate the number of metabolites exchanged between species that coexisted in cocultures outcomes from (A). Edge width represents the number of exchanged metabolites. Exchange networks based on the remaining 10 coculture outcomes are contained in dataset S5. (C) A graphical summary of metabolite exchanges that likely contribute to the coculture outcomes. Bolded metabolites are all inferred causal metabolites that may contribute to the growth of individual species. Gray metabolites represent broadly exchanged growth supporting substrates that are secreted by degraders or exploiters grown on GlcNAc and consumed by scavengers. All remaining secreted metabolites and cross-feeding interactions that are not included in this illustration are contained in datasets S1, S3, and S5. DHMB, 2,3-dihydroxy-2-methylbutanoate.