Spatial Heterogeneity in Electron Acceptor Availability Stimulates Clonal Diversification in Biofilms

In non-laboratory settings bacteria typically reside in matrix-encased biofilm communities. The polymeric biofilm matrix, like other cooperative secretions produced by bacteria, has the potential to be exploited by non-producing bacteria that avoid the energy cost associated with matrix production (i.e. cheaters). Theory, however, predicts that matrix production should be stable against cheating under substrate limitation. Here we show empirically that matrix production by Pseudomonas aeruginosa PA14, a major biofilm-forming pathogen, is favorable under conditions of electron acceptor limitation but not when electron acceptor is abundant. We show that distinct locations within the same biofilm can either select for or against matrix production depending on the local availability of electron acceptor. Our results are consistent with theoretical models, which predict that matrix production allows cell lineages to position themselves favorably within electron acceptor gradients. However, spatial differences in electron acceptor availability within the same biofilm can lead to clonal heterogeneity. By integrating concepts from biofilm engineering and evolutionary biology we suggest a model for the emergence of polyclonality in P. aeruginosa biofilms, a finding relevant for our understanding of persistent infections.