Density of founder cells affects spatial pattern formation and cooperation in Bacillus subtilis biofilms

Abstract

In nature, most bacteria live in surface-attached sedentary communities known as biofilms. Biofilms are often studied with respect to bacterial interactions. Many cells inhabiting biofilms are assumed to express 'cooperative traits', like the secretion of extracellular polysaccharides (EPS). These traits can enhance biofilm-related properties, such as stress resilience or colony expansion, while being costly to the cells that express them. In well-mixed populations cooperation is difficult to achieve, because non-cooperative individuals can reap the benefits of cooperation without having to pay the costs. The physical process of biofilm growth can, however, result in the spatial segregation of cooperative from non-cooperative individuals. This segregation can prevent non-cooperative cells from exploiting cooperative neighbors. Here we examine the interaction between spatial pattern formation and cooperation in Bacillus subtilis biofilms. We show, experimentally and by mathematical modeling, that the density of cells at the onset of biofilm growth affects pattern formation during biofilm growth. At low initial cell densities, co-cultured strains strongly segregate in space, whereas spatial segregation does not occur at high initial cell densities. As a consequence, EPS-producing cells have a competitive advantage over non-cooperative mutants when biofilms are initiated at a low density of founder cells, whereas EPS-deficient cells have an advantage at high cell densities. These results underline the importance of spatial pattern formation for competition among bacterial strains and the evolution of microbial cooperation.

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

“…While single cell based motility is critical during initiation of biofilm development at the air-liquid interface in both Gram-positive and -negative bacteria . However, spatial organization during the development of B. subtilis colony biofilms depends on the density of the bacterial inoculum used to initiate the biofilm 8 .…”

“…Simple microbial systems can be utilized to understand the consequence of spatial structures on the evolution of social interactions 3,4 . Publications in the last 2-3 years using both eukaryotic and prokaryotic model systems highlighted the impact of spatial structures on the stability of cooperation within microbial populations [5][6][7][8] . Additionally, obligate interactions among microbes, e.g.…”

Extracellular matrix structure governs invasion resistance in bacterial biofilms

“…These biofilm properties, in turn, will have ramifications on the evolution of bacterial behavior, for example, investment into local competition versus dispersal (Hanski et al, 2011), and potentially for the development of novel probiotic strategies for enhancing or inhibiting biofilms (Preidis and Versalovic, 2009;Rendueles et al, 2013). Invasion dynamics will also influence the evolutionary stability of extracellular matrix production (Xavier and Foster, 2007;van Gestel et al, 2014). The biofilm matrix is expensive to produce and confers strong fitness benefits, including tolerance to shear forces, environmental toxins and predation by protists and immune cells (Sutherland, 2001;Flemming and Wingender, 2010;Abee et al, 2011;Kovács et al, 2012;Billings et al, 2013).…”

Biofilm formation and toxin production provide a fitness advantage in mixed colonies of environmental yeast isolates

“…Initial cell counts were made in one of two ways: (1) plating 100 μl of culture from the wells, and for mixed colonies either replica‐plating to appropriate antibiotic plates or viewing colonies under a fluorescence stereoscope in order to count the number of colonies of each resistance/color, or (2) imaging 10 μl of culture from the wells with a hemocytometer and differentiating strains from mixed cultures with fluorescence markers. Colonies were started with ~500–1,000 cells, as previous work has shown that starting with a low density can itself generate spatial segregation (van Gestel et al., 2014). …”

“…Despite their vulnerability to individual cheaters, biofilms are ubiquitous and stable. The leading hypothesis for the stability of biofilm communities is the spatial structure: Competition, cooperation, and passive processes like clonal growth can generate patches of related cooperative cells able to outcompete unrelated cells (e.g., (Anderson, Garcia, & Cotter, 2014; van Gestel, Weissing, Kuipers, & Kovacs, 2014; Hallatschek, Hersen, Ramanathan, & Nelson, 2007; Millet et al., 2014; Momeni, Brileya, Fields, & Shou, 2013; Müller, Neugeboren, Nelson, & Murray, 2014; Nadell & Bassler, 2011; Nadell, Foster, & Xavier, 2010; Van Dyken, Müller, Mack, & Desai, 2013; Xavier & Foster, 2007), recently reviewed in detail in ref. (Nadell, Drescher, & Foster, 2016)).…”