Structure-Activity Relationships in Adhesins from Environmental Bacteria

Abstract

The aggregation of bacteria into the form of biofilms is a measure of their success in colonizing biotic and abiotic surfaces. Biofilm communities provide the sheltering bacteria with resistance against biocides like antibiotics, and physical disruption from forces like shearing and abrasion, as well as opportunities to swap beneficial DNA sequences. Bacterial biofilms can be harmful as in tooth plaque or chronic infections, or they can be beneficial as in promoting plant growth by excluding harmful microorganisms. Bacteria use adhesion proteins (adhesins) to initiate the interaction with host surfaces. Characterizing these proteins may provide opportunities to inhibit pathogenic biofilms and reinforce beneficial ones. The purpose of this thesis was to investigate the structure and binding partners of ligand-binding domains from select repeats-in-toxic (RTX) adhesins, and to find novel domains with similar functions. The examples pursued here include: 1) The large adhesion protein (Lap) from the rhizobacterium Pseudomonas fluorescens, which contains a peptide-binding domain called vWFA domain. The domain, being different from the orthologs in eukaryotes, contains an extra insert region adjacent to the metal-binding site. Structural alignment with vWFAs from other bacterial species indicated that this region might affect ligand selection. 2) A sugar-binding PA14 domain was recognized in Pseudomonas fluorescens Lap as a highly conserved ortholog of the one from Marinomonas primoryensis ice-binding protein. To establish the importance of the D-cis-D motif during biofilm formation, the double aspartates in the binding site were mutated to alanines, which eliminated the domain’s ability to bind glucose. Lastly, 3) The large adhesion protein from the oil-degrading bacterium Marinobacter hydrocarbonoclasticus contains a second sugar-binding PA14_2 domain besides the previously characterized PA14 domain. A recombinant version of the second ligand-binding domain was produced in E. coli. Its elution during Superdex 75 size-exclusion chromatography was retarded, suggesting it might have a sugar-binding ability. Glycan array analysis showed that the domain had a preference for binding carbohydrates extracted from Proteus mirabilis. This result might help identify the type of organisms on which the oil-degrading bacteria form biofilms.