Graduation Date

Spring 5-7-2016

Document Type


Degree Name

Doctor of Philosophy (PhD)


Pathology & Microbiology

First Advisor

Kenneth W. Bayles


Chronic biofilm-related infections caused by the human pathogen Staphylococcus aureus often lead to significant increases in morbidity and mortality in both hospital- and community-associated settings. Typically, S. aureus biofilm development occurs in three stages: 1) attachment, 2) tower maturation, and 3) dispersal. Here, utilizing BioFlux1000 time-lapse microscopy we have expanded upon these fundamental stages of biofilm development and also unveiled and characterized two additional stages (multiplication and exodus).

The attachment and multiplication stages were shown to be protease sensitive but independent of most cell surface-associated proteins. Following multiplication, an exodus of the biofilm population that followed the transition of the biofilm to DNase I sensitivity was demonstrated. Furthermore, disruption of the gene encoding staphylococcal nuclease (nuc) abrogated this exodus event, causing hyper-proliferation of the biofilm and disrupting tower development. Prior to exodus, cells carrying a Pnuc::gfp promoter fusion demonstrated Sae-dependent expression, but only in a subpopulation of cells. Additionally, we also determined that other Sae-regulated genes demonstrated unique Sae-dependent stochastic expression patterns. Collectively, these results suggest the presence of a Sae-controlled nuclease-mediated exodus of a biofilm subpopulation that is required for tower development as well as controlling the stochastic expression of Sae-regulated factors.

The cidABC and lrgAB operons have previously been shown to play specific roles in controlled cell death and release of extracellular DNA (eDNA) during biofilm maturation. Although the exact mechanisms controlling the cid and lrg operons have yet to be completely elucidated, the expression of the operons is dependent on altered metabolic cues as a result of overflow metabolism. We hypothesized that the differential expression of the cid and lrg operons within a biofilm is a function of the metabolic heterogeneity found within different biofilm microenvironments. Time-lapse epifluorescent images indicate that expression of these operons is specific to distinct regions of a growing biofilm. Additionally, these results revealed the existence of different tower types, possibly reflecting their different functional roles in development. Altogether, nuclease-mediated eDNA degradation modulates the biofilm to produce two distinct towers that there are both spatially and temporally different between compared to the rest of the biofilm.

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Bacteriology Commons