| Resolving the genetic and mechanistic bases of complex biological behaviors remains a central challenge in the post-genomic era. Among these is the emergence of interspecies cooperation, a feature common across levels of biological organization. Of the numerous examples afforded by nature, microbes arguably provide the greatest ability to connect underlying genotypes to cooperative phenotypes.;Using an engineered bacterial consortium, we repeatedly evolved cooperation and tested how interspecies dynamics impact the predictability of evolution. Eight Salmonella enterica serovar Typhimurium strains evolved methionine excretion sufficient to support growth of an Escherichia coli methionine auxotroph, from whom they required excreted growth substrates. Non-synonymous mutations in metA, encoding homoserine trans-succinylase, were detected in each evolved S. enterica methionine cooperator, and were shown to be necessary for cooperative consortia growth. Despite this genetic parallelism, these metA alleles gave rise to a wide range of phenotypic diversity in terms of individual versus group benefit. The cooperators with the highest methionine excretion permitted nearly two-fold faster consortia growth and supported the highest fraction of E. coli, yet interestingly also had the slowest individual growth rates compared to less cooperative strains.;The two-step selective protocol used to evolve these cooperators, however, raised questions about historical contingency, which is believed to play a large role in shaping evolution. Without initial selection for resistance to end-product transcriptional inhibition, S. enterica struggled to evolve cooperation. Selection for resistance to a toxic methionine analog, ethionine, enabled more efficient evolution of cooperation by S. enterica in a process that required two adaptive mutations. When bacteria must overcome multiple levels of metabolic repression to excrete costly compounds, gene interactions like epistasis may limit adaptive strategies. In this consortium, epistasis between metJ and metA adaptive mutations suggests microbes undergoing de novo evolution of cooperation face similar challenges.;Knowing how previous selective pressures and interspecies dynamics impact adaptive variation at the genetic, phenotypic, and ecological levels will better constrain our ability to predict complex microbial community behavior from the genotypes or phenotypes of the strains within them. |
