Investigation of the role of Caulobacter crescentus surface structures in surface attachment and biofilm formation

Caulobacter crescentus has several surface structures including pili, a flagellum and a holdfast, which are involved in attachment to surfaces and biofilm formation, but their roles in the different stages of biofilm maturation are still unclear. Our studies indicate that in a static or dynamic environment, pili (pilA) and flagellum motor ( motB) mutants were greatly impaired in all stages of surface attachment and biofilm formation, and that a flagellum hook (flgE) mutant formed biofilm faster and more robustly than the wild-type strain in a hydrodynamic environment. Our results reveal that the increased surface attachment of the hook mutant could be due to the earlier secretion of the holdfast adhesive.;C. crescentus cells are covered by a proteinacious surface layer (S-layer) but it was not know if it is involved in attachment and biofilm formation. An S-layer (rsaA) mutant grown statically formed a more robust biofilm than wild-type, and removal of the S-layer by mutation in a non-adherent holdfast mutant partially restored the ability to attach to surfaces and to form biofilms. These results suggest that exposure of the surface lipopolysaccharide (LPS) by removal of the S-layer caused non-specific surface attachment through the LPS. In contrast, the rsaA mutant grown in a shaking environment produced less biofilm than when grown under static conditions after 12 h, indicating that the S-layer plays a role in biofilm integrity. A manB deletion resulting in loss of smooth LPS was used to determine if the exposed rough LPS of an S-layer mutant is responsible for the increase in surface attachment. In a static environment at 24 h, biofilm formation increased in the manB mutant as compared to wild-type. However, the manB mutant never made as much biofilm when compared to the rsaA mutant. After 24 h, the manB mutant biofilm was very unstable suggesting that LPS is involved in the robust biofilm of the rsaA mutant.