Authors: Robin Tecon, Annemieke van der Wal, Mitja Remus-Emsermann and Johan Leveau. Abstract: Most microbial habitats are characterized by physical, chemical, and biological conditions that differ significantly along micrometer distances. This environmental heterogeneity at the scale of individual microbes may account for much of the nonrandom distribution of microorganisms observed in natural settings. Here we report on an individual-based approach that combines spatially explicit modeling and bioreporter-driven experimentation to improve our basic understanding of the impact of small-scale heterogeneity on the bacterial colonization of surfaces. We used bioreporters of Erwinia herbicola 299R tagged with the green fluorescent protein (GFP) in two ways. One type of bioreporter constitutively expressed GFP which allowed for easy visualization of the location of bacteria in relation to one another. In the second type of bioreporter, preformed GFP was diluted from cells as they divided, rendering GFP content of daughter cells an inverse function of the reproductive success of their mothers. These bioreporter cells were inoculated on a defined agarose surface and analyzed by fluorescence microscopy to measure bacterial growth and colony formation during incubation. E. herbicola cells all grew at the same rate on the surface of the gel medium, regardless of the bacterial spatial density. This suggests that under these conditions, growth of bacteria was not influenced by the presence of others nearby. Fluorescence intensity and surface area measurements of the reproductive success were in good correlation. The pattern simulation matched the experimental data when it introduced initial variations in the cell cycle of colonizers. This approach of validating bioreporter output in simple environments with defined complexity are essential for our ability to explain their performance in more complex environments, such as the natural habitat of E. herbicola 299R, i.e. the plant leaf surface
