| Many species within the alpha-proteobacteria can synthesize a polarly localized polysaccharide structure called holdfast. Among them, Caulobacter crescentus holdfast has been well established as a polarly-localized, strong bio-adhesive, and the production of holdfast is essential for surface attachment and biofilm formation. The hfs (hold fast synthesis) gene cluster was previously found to be involved in holdfast synthesis and export. Asticcacaulis biprosthecum is closely related with C. crescentus, and it also harbors a holdfast at the flagellar pole. Several adhesion mutants were isolated and characterized with transposon insertions in the hfs gene cluster of A. biprosthecum. Genomic analysis demonstrates that the A. biprosthecum hfs gene cluster has the same composition and organization as the C. crescentus hfs cluster. The hfs mutations in A. biprosthecum also have similar adhesion deficiency as those of C. crescentus. Interestingly, the holdfasts of the A. biprosthecum and C. crescentus hfsH mutants have impaired anchoring to cell body. Further fluorescence microscopy quantification of C. crescentus hfsH mutant holdfasts revealed that they are smaller than those of wildtype, and the shed holdfasts are even smaller than the attached ones. The super resolution microscopy (OMX) detected "fiber-like" material from the C. crescentus DeltahfsH mutant holdfasts attached to a glass coverslip, and the attached holdfasts from the Delta hfsH mutant were less resistant to shear force than those of wildtype in the ONIX microfluidics apparatus. These data suggest that the C. crescentus DeltahfsH mutant holdfasts have lower adhesiveness and cohesiveness than those of wildtype. Site-directed mutagenesis at the predicted HfsH enzymatic residue of C. crescentus results in the same phenotype as the clean deletion mutant, which supports the function of HfsH as a polysaccharide deacetylase essential for adhesive and cohesive properties of holdfast.;The localization and function of HfsD/A/B were also analyzed both in vitro and in vivo using biochemical and microscopic approaches. HfsD is identified as an outer membrane protein, and exhibits polar localization that is cell cycle dependent. Proper HfsA and HfsD cell envelope localization requires the presence of all holdfast export proteins, and is critical for holdfast production. The overexpression of HfsA, HfsD, or HfsB in DeltahfsB and DeltapodJ mutants rescued the localization of HfsA and HfsD, and holdfast synthesis/surface adhesion deficiencies. Interestingly, in these cases, the restored holdfasts were found to be randomly attached to the cell surface, but often co-localized with HfaB and HfsD foci. Therefore, it is hypothesized that overexpression of holdfast export proteins allows ectopic aggregation of a holdfast biogenesis/anchoring complex by locally surpassing a concentration threshold necessary for protein secretion into the outer membrane and holdfast synthesis. |
