Non-disruptive matrix turnover is a conserved feature of biofilm aggregate growth in paradigm pathogenic species

Bacteria form multicellular aggregates called biofilms. A crucial component of these aggregates is a protective matrix that holds the community together. Biofilm matrix composition varies depending upon bacterial species but typically includes exopolysaccharides (EPS), proteins, and extracellular DNA. Pseudomonas aeruginosa is a model organism for the study of biofilms, and in non-mucoid biofilms, it uses the structurally distinct EPS Psl and Pel, the EPS-binding protein CdrA, and eDNA as key matrix components. An interesting phenomenon that we and others have observed is that the periphery of a biofilm aggregate can be EPS-rich and contain very few cells. In this study, we investigated two possible models of assembly and dynamics of this EPS-rich peripheral region: (i) newly synthesized EPS is inserted and incorporated into the existing EPS-rich region at the periphery during biofilm aggregate growth or (ii) EPS is continuously turned over and newly synthesized EPS is deposited at the outermost edge of the aggregate. Our results support the latter model. Specifically, we observed that new EPS is continually deposited at the aggregate periphery, which is necessary for continued aggregate growth but not aggregate stability. We made similar observations in another paradigm biofilm-forming species, Vibrio cholerae. This pattern of deposition raises the question of how EPS is retained. Specifically, for P. aeruginosa biofilms, the matrix adhesin CdrA is thought to retain EPS. However, current thinking is that cell-associated CdrA is responsible for this retention, and it is not clear how CdrA might function in the relatively cell-free aggregate periphery. We observed that CdrA is enzymatically degraded during aggregate growth without negatively impacting biofilm stability and that cell-free CdrA can partially maintain aggregation and Psl retention. Overall, this study shows that the matrix of P. aeruginosa biofilms undergoes both continuous synthesis of matrix material and matrix turnover to accommodate biofilm aggregate growth and that cell-free matrix can at least partially maintain biofilm aggregation and EPS localization. Furthermore, our similar observations for V. cholerae biofilms suggest that our findings may represent basic principles of aggregate assembly in bacteria.