Engineering Cooperation in an Anaerobic Coculture

Abstract

Over the past century, microbiologists have studied organisms in pure culture, yet it is becoming increasingly apparent that the majority of biological processes rely on multispecies cooperation and interaction. While little is known about how such interactions permit cooperation, even less is known about how cooperation arises. To study the emergence of cooperation in the laboratory, we constructed both a commensal community and an obligate mutualism using the previously noninteracting bacteria Shewanella oneidensis and Geobacter sulfurreducens Incorporation of a glycerol utilization plasmid (pGUT2) enabled S. oneidensis to metabolize glycerol and produce acetate as a carbon source for G. sulfurreducens, establishing a cross-feeding, commensal coculture. In the commensal coculture, both species coupled oxidative metabolism to the respiration of fumarate as the terminal electron acceptor. Deletion of the gene encoding fumarate reductase in the S. oneidensis/pGUT2 strain shifted the coculture with G. sulfurreducens to an obligate mutualism where neither species could grow in the absence of the other. A shift in metabolic strategy from glycerol catabolism to malate metabolism was associated with obligate coculture growth. Further targeted deletions in malate uptake and acetate generation pathways in S. oneidensis significantly inhibited coculture growth with G. sulfurreducens The engineered coculture between S. oneidensis and G. sulfurreducens provides a model laboratory system to study the emergence of cooperation in bacterial communities, and the shift in metabolic strategy observed in the obligate coculture highlights the importance of genetic change in shaping microbial interactions in the environment.IMPORTANCE Microbes seldom live alone in the environment, yet this scenario is approximated in the vast majority of pure-culture laboratory experiments. Here, we develop an anaerobic coculture system to begin understanding microbial physiology in a more complex setting but also to determine how anaerobic microbial communities can form. Using synthetic biology, we generated a coculture system where the facultative anaerobe Shewanella oneidensis consumes glycerol and provides acetate to the strict anaerobe Geobacter sulfurreducens In the commensal system, growth of G. sulfurreducens is dependent on the presence of S. oneidensis To generate an obligate coculture, where each organism requires the other, we eliminated the ability of S. oneidensis to respire fumarate. An unexpected shift in metabolic strategy from glycerol catabolism to malate metabolism was observed in the obligate coculture. Our work highlights how metabolic landscapes can be expanded in multispecies communities and provides a system to evaluate the evolution of cooperation under anaerobic conditions.

Keywords: Geobacter; Shewanella; anaerobic respiration; metabolism.

FIG 1

Metabolic interactions enabling commensal coculture growth of S. oneidensis/pGUT2 and G. sulfurreducens. S. oneidensis oxidizes glycerol via an engineered glycerol utilization pathway (GlpF, glycerol facilitator; GlpK, glycerol kinase; GlpD, glycerol-3-P dehydrogenase; TpiA, triose phosphate isomerase) and secretes acetate that is used as a carbon source by G. sulfurreducens. Cross-feeding of acetate from S. oneidensis/pGUT2 to G. sulfurreducens is represented by the black arrow. Both organisms couple metabolic oxidation reactions to the respiration of fumarate (FccA and Frd represent fumarate reductase in S. oneidensis and G. sulfurreducens, respectively).

FIG 2

Growth of S. oneidensis/pGUT2 and G. sulfurreducens alone and in coculture in basal coculture medium (CM) containing 7 mM glycerol (G) and/or 60 mM fumarate (F) as indicated. (A) Growth measured as OD₆₀₀ of the S. oneidensis/pGUT2-G. sulfurreducens coculture in CMGF (blue circles), CMG (black squares), or CMF (gray triangles) medium compared to that of S. oneidensis/pGUT2 (green triangles) or G. sulfurreducens (red diamonds) grown singly in CMGF. Growth was not observed for S. oneidensis/pGUT2 or G. sulfurreducens cultured alone in CMG or CMF, and data were removed for graph clarity. (B) Growth monitored as CFU in CMGF medium for S. oneidensis/pGUT2 (green circles) and G. sulfurreducens (red squares) grown alone (open symbols) or in coculture (closed symbols). Reported values are averages for triplicate experiments with error represented as standard error of the mean (SEM).

FIG 3

Metabolite profiles in (A) S. oneidensis/pGUT2-G. sulfurreducens cocultures, (B) S. oneidensis/pGUT2 only, and (C) G. sulfurreducens only cultures in CMGF medium containing 7 mM glycerol and 60 mM fumarate. Metabolites include glycerol (green triangles), acetate (red squares), fumarate (blue circles), and succinate (purple inverted triangles). Reported values are for three independent experiments with error represented as SEM. Error bars associated with the above data were small and hence are masked due to the symbol size used.

FIG 4

Obligate growth of S. oneidensis ΔfccA/pGUT2 and G. sulfurreducens in basal coculture medium (CM) containing 7 mM glycerol and 60 mM fumarate (CMGF, closed symbols) or coculture medium supplemented only with 60 mM fumarate (CMF, open symbols). (A) Growth measured as OD₆₀₀ for S. oneidensis ΔfccA/pGUT2 and G. sulfurreducens in coculture (blue) and for S. oneidensis ΔfccA/pGUT2 (green) and G. sulfurreducens (red) cultured alone. (B) Growth measured as CFU for S. oneidensis ΔfccA/pGUT2 (green) and G. sulfurreducens (red) in coculture in CMGF (closed symbols) and CMF (open symbols) media. Reported values are averages from triplicate experiments with error bars representing SEM.

FIG 5

Metabolite profiles in S. oneidensis ΔfccA/pGUT2-G. sulfurreducens cocultures in (A) CMGF medium or (B) CMF medium, S. oneidensis ΔfccA/pGUT2 cultured alone in (C) CMGF medium or (D) CMF medium, and G. sulfurreducens cultured alone in (E) CMGF medium or (F) CMF medium. Metabolites include glycerol (green), fumarate (blue), malate (orange), and succinate (purple). Media contained 7 mM glycerol and/or 60 mM fumarate as indicated. Reported values represent mean ± SEM metabolite concentrations (mM) measured for three independent experiments.

FIG 6

Obligate growth of S. oneidensis ΔfccA Δpta/pGUT2 and G. sulfurreducens in basal coculture medium (CM) containing 7 mM glycerol and 60 mM fumarate (CMGF, closed symbols) or coculture medium supplemented only with 60 mM fumarate (CMF, open symbols). (A) Growth measured as OD₆₀₀ for the S. oneidensis ΔfccA Δpta/pGUT2-G. sulfurreducens coculture (blue) and for S. oneidensis ΔfccA Δpta/pGUT2 (green) and G. sulfurreducens (red) cultured singly. (B) Growth measured as CFU/ml media for S. oneidensis ΔfccA Δpta/pGUT2 (green) and G. sulfurreducens (red) cocultures. Reported values are averages for triplicate experiments with error bars representing SEM.

FIG 7

Metabolite profiles in S. oneidensis ΔfccA Δpta/pGUT2-G. sulfurreducens cocultures in (A) CMGF medium or (B) CMF medium and in S. oneidensis ΔfccA Δpta/pGUT2 cultured alone in (C) CMGF medium or (D) CMF medium. Metabolites include glycerol (green), fumarate (blue), malate (orange), and succinate (purple). Media contained 7 mM glycerol and/or 60 mM fumarate as indicated. Reported values represent mean ± SEM metabolite concentrations (mM) measured for three independent experiments.

FIG 8

Obligate growth of S. oneidensis ΔfccA ΔmaeB ΔsfcA ΔpckA/pGUT2 and G. sulfurreducens in basal coculture medium (CM) containing 7 mM glycerol and 60 mM fumarate (CMGF, closed symbols) or coculture medium supplemented with only 60 mM fumarate (CMF, open symbols). (A) Growth measured as OD₆₀₀ for the S. oneidensis ΔfccA ΔmaeB ΔsfcA ΔpckA/pGUT2-G. sulfurreducens coculture (blue) and for S. oneidensis ΔfccA ΔmaeB ΔsfcA ΔpckA/pGUT2 (green) and G. sulfurreducens (red) cultured singly. (B) Growth measured as CFU/ml media for S. oneidensis ΔfccA/pGUT2 (green) and G. sulfurreducens (red) cocultures. Reported values are averages for triplicate experiments with error bars representing SEM.

FIG 9

Metabolite profiles in S. oneidensis ΔfccA ΔmaeB ΔsfcA ΔpckA/pGUT2-G. sulfurreducens cocultures in (A) CMGF medium or (B) CMF medium and in S. oneidensis ΔfccA ΔmaeB ΔsfcA ΔpckA/pGUT2 cultured alone in (C) CMGF medium or (D) CMF medium. Metabolites include glycerol (green), fumarate (blue), malate (orange), and succinate (purple). Media contained 7 mM glycerol and/or 60 mM fumarate as indicated. Reported values represent mean ± SEM metabolite concentrations (mM) measured for three independent experiments.

FIG 10

Obligate growth of S. oneidensis ΔfccA and G. sulfurreducens in basal coculture medium (CM) containing 7 mM glycerol and 60 mM fumarate (CMGF, closed symbols) or coculture medium supplemented only with 60 mM fumarate (CMF, open symbols). (A) Growth measured as OD₆₀₀ for the S. oneidensis ΔfccA-G. sulfurreducens coculture (blue) and for S. oneidensis ΔfccA (green) and G. sulfurreducens (red) cultured singly. (B) Growth measured as CFU/ml media for S. oneidensis ΔfccA (green) and G. sulfurreducens (red) cocultures. Reported values are averages for triplicate experiments with error bars representing SEM.

FIG 11

Metabolite profiles in S. oneidensis ΔfccA-G. sulfurreducens cocultures in (A) CMGF medium or (B) CMF medium or in S. oneidensis ΔfccA cultured alone in (C) CMGF medium or (D) CMF medium. Metabolites include glycerol (green), fumarate (blue), malate (orange), and succinate (purple). Media contained 7 mM glycerol and/or 60 mM fumarate as indicated. Reported values represent mean ± SEM metabolite concentrations (mM) measured for three independent experiments.