Engineering cooperation in an anaerobic co-culture

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 non-interacting 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 co-culture. In the commensal co-culture, 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 co-culture with G. sulfurreducens to an obligate mutualism where neither species could grow in absence of the other. A shift in metabolic strategy from glycerol catabolism to malate metabolism was associated with obligate co-culture growth. Further targeted deletions in malate uptake and acetate generation pathways in S. oneidensis significantly inhibited co-culture growth with G. sulfurreducens. The engineered co-culture 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 co-culture 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 co-culture 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 co-culture 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 co-culture, 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 co-culture. Our work highlights how metabolic landscapes can be expanded in multi-species communities and provides a system to evaluate the evolution of cooperation under anaerobic conditions.