Molecular mechanisms and benefits of phenotypic heterogeneity in Sinorhizobium meliloti populations

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Phenotypic heterogeneity is prevalent in the microbial world, especially in biofilms. We describe a case using colonies of the alpha-proteobacterium bacterium Sinorhizobium meliloti. A sub-population strongly expresses genes specific for exopolysaccharide (EPS) production, while their genetically identical neighbors exhibit either very little or no expression. Although the genetic pathways controlling EPS are relatively well understood, such as quorum sensing, little is known about how multiple phenotypes with respect to EPS production arise. To understand this phenomenon, we have used an interdisciplinary approach, integrating mathematical modeling, genetic engineering, live cell imaging, and data processing analyses. The approach relies upon a triple-reporter construct which is capable of carrying three different promoters, each fused to a dedicated fluorescent reporter gene. This provides a method whereby the growth of a colony from a single cell can be monitored, and each individual can be systematically classified with regards to its ancestral lineage and its contributor status. We classify each individual into one of three classes: non-contributor, weak contributor, and strong contributor.On the basis of this platform, we outline a research program to address the question of how heterogeneity arises in the regulatory circuitry controlling EPS production. The program focuses on several aspects of regulatory circuits essential for bi-stability. One is the presence of mechanisms capable of generating a heterogenic signal (e.g., hemi-methylation of promoter regions, and the stress response), and another is mechanisms capable of heterogenic signal amplification (e.g., positive feedback loops). Both features have been found in the regulatory circuitry controlling EPS production in S. meliloti. These will be integrated in discriminatory theoretical models which are testable in the laboratory. Another important question is the biological impact of phenotypic heterogeneity. To answer this, we make use of our recent work which compares a mutant (displaying a non-contributor phenotype) to the wild type. The mutant grew faster and was more motile, while the wild type was better at survival of harsh physical conditions. In this program, we propose a method to obtain populations that are selected for a single class by fusing the promoters controlling EPS production to positive and negative selection cassettes. This will allow the conditional enrichment of single-class populations without further genetic perturbation and allow characterization of the contributor and non-contributor classes. Finally, we ask the question of how the cooperative/contributor phenotypes of S. meliloti can be favored in the context of evolution and develop a model addressing this question.