Archaeal type IV pili stabilize Haloferax volcanii biofilms in flow

Biofilms represent a prevalent lifestyle of unicellular organism that confers protection to external challenges. The mechanisms by which archaea form biofilms are however not entirely clear. H. volcanii is an extremely halophilic euryarchaeon that commonly colonizes salt crust surfaces. H. volcanii produces long and thin appendages called type IV pili that are known to play a function in surface attachment and biofilm formation in archaea and bacteria. Here, we used biophysical experiments to identify critical function of type IV pili in the mechanical integrity of H. volcanii biofilms. Using interferometric scattering microscopy (iSCAT) to non-invasively visualize T4P in live cells, we find that piliation varies across mutants expressing single pilin isoforms. Using microfluidic experiments, we found that the adhesive strength of these mutants correlates with their extent of piliation. We found that in flow, H. volcanii forms clonal biofilms that extend in three dimensions. Expression of PilA2, a single pilin isoform, is sufficient to maintain normal levels of piliation and form biofilms with a structure indistinguishable from WT. Furthermore, we found that fluid flow is a crucial determinant of biofilm integrity: in the absence of flow, biofilms lose cohesion and tend to disperse in a density-dependent manner. Overall, our results demonstrate that T4P-surface and possibly T4P-T4P interactions promote biofilm formation and integrity, and that flow is a crucial ingredient regulating archaeal biofilm formation. ### Competing Interest Statement The authors have declared no competing interest.