Biophysical Constraints Dictate the Stability of Social Traits in Pseudomonas aeruginosa Aggregates

The maintenance of cooperative behaviors within microbial populations remains an evolutionary puzzle, particularly in spatially structured environments like those found in chronic infection. In Pseudomonas aeruginosa, quorum sensing (QS) directs the production of costly public goods that are vulnerable to exploitation by non-producing cheaters. However, the biophysical mechanisms that stabilize this cooperation in complex environments remain poorly understood. Here, we elucidate how the biophysical properties of the bacterial cell surface govern the micron-scale architecture of bacterial aggregates to promote or constrain the stability of cooperation. Using a polymer-structured growth environment, we demonstrate that cell surface hydrophobicity acts as a primary determinant of spatial organization, where hydrophilic wild-type cells assemble into "stacked" aggregates, whereas hydrophobic O-specific antigen-deficient cells form dense "clumps". In mixed populations, distinct surface properties exhibit phase-separation-like immiscibility, segregating the cells at the micron scale. We show that this segregation directly suppresses cheater fitness by physically sequestering cooperative clusters. Specifically, hydrophobic QS-deficient cells were effectively excluded by hydrophilic QS cooperators, limiting their access to public goods. Furthermore, invasion experiments revealed that hydrophilic cells are inherently fitter, capable of unidirectionally invading populations with a hydrophobic cell surface and independent of social dynamics. Conversely, the need to gain access to nutrient resources can fine-tune these barriers, enabling hydrophilic QS-deficient cells to cluster proximally to hydrophobic cooperators. These findings establish that bacterial cell surface traits impact strongly on microbial social interactions and offer new insights into the maintenance and loss of cooperative traits in chronic infections such as those found in chronic lung infections.