Inferring Bacterial Interspecific Interactions from Microcolony Growth Expansion

Interactions between species are thought to be crucial for modulating their growth and behaviour within communities, and determinant for the emergence of community functions. Several different interaction concepts exist, but there is no consensus on how interactions should be quantified and integrated in community growth theory. Here we expand on existing concepts of real-time measurements of pure culture microcolony growth to develop and benchmark coculture microcolony experiments, and show how these can both parametrize growth kinetic and interspecific interaction effects. We follow surface growth by time-lapse microscopy of fluorescently tagged Pseudomonas putida and Pseudomonas veronii under substrate competition with succinate, or under substrate indifference with D-mannitol and putrescine. Monoculture-grown microcolonies showed substrate concentration dependent expansion rates as expected from Monod relations, whereas individual microcolony yields were strongly dependent on densities and spatial positioning of founder cells. Maximum specific growth rates in cocultures under substrate competition were diminished by ca. 15%, which was seeding-density independent. The collective P. putida population dominated growth over that of P. veronii, but with 27% yield loss under competition compared to monoculture growth; and 90% for that of P. veronii. Incidental local reversal of competition was observed where P. veronii microcolonies profited at the detriment of P. putida, and between 9 and 43% of P. veronii microcolonies grew bigger than expected from bulk competition, depending on seeding density. Simulations with a cell-agent Monod surface growth model suggested that colony expansion rate decrease in competitive coculture is caused by metabolite cross-feeding, which was supported by exometabolite analysis during and after growth of the strains on their individual or swapped supernatant. Coculture microcolony growth experiments thus provide a flexible platform for analysis of kinetic and interspecific interactions, expanding from individual microcolony phenotypic effects to averaged behaviour across all microcolony pairs. The system in theory is scalable to follow real-time growth of multiple species simultaneously into communities.