Doctoral Dissertations

Date of Award

12-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Todd B. Reynolds

Committee Members

Jeffery Becker, Albrecht Von Arnim, Andreas Nebenfuhr

Abstract

Biofilms are a mode of growth where aggregated cells adhere to a foreign surface and grow as a complex community. Biofilms have found wide utility in commercial industries, however infections caused by biofilms in clinical settings are a major cause of concern. Understanding molecular details of biofilm formation could help in exploitation or elimination efforts.

We utilize Saccharomyces cerevisiae as a model system to study biofilm formation. S. cerevisiae strain belonging to genetic background Σ [sigma] 1278b is capable of forming biofilms, on low density (0.3%) agar media. When grown at 25°[degree] C for 5 days, it develops into an elaborate floral shaped biofilm. The biofilm can be structurally differentiated into a central wrinkly part called hub, and a peripheral smooth part called rim. A flocculin family surface protein Flo11p, known to be essential for the phenotypes of adhesion and invasive growth, is also important for biofilm formation. We identified that certain vacuolar protein sorting (VPS) proteins don’t affect Flo11p expression and yet were defective in biofilm formation. Thus showing that the phenotypes requiring Flo11p (invasive growth and adhesion) are genetically separable from the phenotype of biofilm formation.

We propose a model showing the existence of a putative biofilm pathway involving endosomal Multivesicular body (MVB) pathway, which affects biofilm formation without causing any defects in Flo11p expression or localization. We further identified that the cell wall integrity (CWI) pathway is partially involved in the biofilm pathway, and supposedly affects biofilm formation by causing defects in cell wall structure.

Although there is no detectable difference in Flo11p expression levels between the rim and hub cells within the biofilm, they are very distinct in appearance and also manifest differences in adhesion. What molecular markers contribute to these differences, however is not yet known. Using RNA-Seq, a high throughput sequencing method, differential expression levels of genes between the rim and hub was obtained. Analysis of the genes revealed the presence of a carbohydrate, named chitosan, in the hub. Further tests showed that though chitosan is not essential for biofilm formation, it plays a protective role against cell wall stressing agents in biofilms.

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