Kinetic Analysis of S. aureus Biofilm Regulation by Using Combined Imaging and Genome-Wide Approaches

Staphylococcus aureus is a ubiquitous gram-positive bacterium, able to exist on inanimate objects as well as in a variety of niches, such as the skin, mucous membranes, blood, or bones. During its lifetime, a single bacterium can travel from one location to another, exposing the bacteria to multiple environments. These transitions are very likely to require quick switches from a unicellular planktonic lifestyle to settle multicellular communities commonly referred to as biofilms. In this application, we have set the following objectives:

• (i) further characterize the three mutants (SA0701, codY, and SA1885) using different physiological and biochemical assays
• (ii) perform morphological analysis of biofilm development and maturation using confocal light scanning microscopy of fluorescently-labeled S. aureus,
• (iii) correlate morphologic stages with defined patterns of gene expression,
• ((iv) replicate the 38 mutants by constructing deletion mutants and their complemented isogenic counterparts in the permissive ?hsdR UAMS-1 background,
• (v) establish a model of dynamic biofilm, (vi) characterize the metabolic conditions of the 38 biofilm-deficient mutants by genome-wide transcription profiling, and
• (vii) quantify the genetic diversity and study mutations that are selected during biofilm maturation.

In summary, molecular and genetic tools developed during previous grant applications will be applied to characterize in details a subset of genes involved in S. aureus biofilm development. We will also address the general question whether biofilm formation represents a developmental program of coordinated gene expression and study the genetic diversification and selection shaping biofilm evolution.

Keywords : Staphylococcus aureus biofilms, host-pathogen interactions, foreign-body infections, mRNA messenger profiling, microarrays, gene regulation, transcriptomics, proteomics, confocal imaging