Bacterial conjugation can restructure biofilms and increase their resilience while constraining host cell dispersal

Bacterial conjugation enables the horizontal transfer of plasmids that often carry genes influencing host physiology and behavior. In spatially structured biofilms, where many bacteria live in close proximity, conjugation can significantly alter both genetic and physical community composition. Here, we use a microfluidic system and fluorescence microscopy to track the transmission of the F-like plasmid pED208 within Escherichia coli biofilms, differentiating invading plasmid donors, transconjugants, and plasmid-free cells at high resolution. We find that conjugation within established resident biofilms is efficient until cell density reaches a threshold associated with high extracellular matrix secretion. Strikingly, plasmid-encoded conjugative pili also enable matrix-deficient cells to aggregate into dense biofilms, promoting the formation of multi-strain and multispecies cell clusters. This restoration of biofilm architecture increases antibiotic and phage tolerance but comes at the cost of altering dispersal dynamics: plasmid-bearing cells disperse less readily than plasmid-free cells, creating a trade-off between local advantage and distal spread. Our findings indicate that conjugative pilus-mediated adhesion incurs a fitness trade-off, compacting biofilm structure and thereby conferring enhanced antibiotic and phage tolerance while reducing the spread of plasmid carriers over larger spatial scales.