Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Joel Weadge

Advisor Role

Supervisor

Abstract

Biofilms are a growing concern in the medical field due to their increased resistance to antibiotics. When found in a biofilm, bacteria can have antibiotic resistance 10-1000 times that of their planktonic counterparts. Therefore, it is important to study the formation of biofilms. Cellulose biofilms are formed by Enterobacteriaceae, such as many Escherichia coli and Salmonella spp. strains. Biofilms provide these species with benefits including antimicrobial protection, development of bacterial communities, promotion of DNA exchange, uptake of nutrients, and, in the case of cellulose biofilms, immune system evasion. Cellulose biofilms are controlled by the Bacterial cellulose synthesis (Bcs) complex located at the cell membrane of bacteria able to form cellulose-based biofilms. Proteins, BcsA and BcsB, have been characterized for cellulose synthesis, however, cellulose export has yet to be described. BcsC is believed to play a role in this export process due to its homology to other polysaccharide export proteins in the alginate and poly β-1,6-GlcNAc (PGA) systems. Herein, a series of bioinformatics analysis was performed that supported the hypothesis that BcsC consists of an outer membrane β-barrel connected to a periplasmic tetratricopeptide repeat (TPR) region and that these two regions play different roles in the export process. To begin addressing this hypothesis, the research focused on the structure-function characterization of these regions of BcsC. While practical quantities of the β-barrel region could be purified, this region proved to be recalcitrant to folding into its native state following purification. However, high yields of all TPR constructs were obtained and subjected to further analyses. Circular dichroism studies confirmed our bioinformatics analyses that the secondary structure of the TPR constructs have a predominantly α-helical content. This technique also provided preliminary evidence that there are structural changes upon binding of the TPR to soluble carboxymethyl cellulose (CMC). Intrinsic fluorescence spectroscopy quenching results further demonstrated that the TPR region has a single binding site along with high KD values (ex. 416 µM for the longest construct) for carboxymethyl cellulose. These results were further confirmed with an Avicel insoluble substrate binding assay which also demonstrated that binding of the TPR to cellulose occurred across a biologically relevant pH range (pH 6-8) and that the majority of the binding may be due to an N-terminal portion of the TPR region (amino acids 24-342). Thus, this collective evidence supports that the TPR region of BcsC plays an integral role in the transport of cellulose polymers across the bacterial cell wall into the external environment where biofilm formation can occur. Future studies regarding BcsC would benefit in investigating potential protein-protein interactions with periplasmic proteins, such as BcsG, as well as profiling the β-barrel domain.

Convocation Year

2015

Convocation Season

Fall

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