AbstractRecent studies of the expression of the Staphylococcus aureus cid and lrg operons during biofilmdevelopment have demonstrated the existence of distinct metabolic microniches that are proposed to servedifferent biological roles. Unfortunately, a critical barrier to understanding these potential roles is thedifficulty in separating the microniche cells from other regions of the biofilm so that they can be subjected tovarious analytical methods. Thus, the overall objective for this project is to apply cellular separationtechniques to isolate the microniche cells and then subject them to comprehensive transcriptomic analysesto gain a better understanding of their basic characteristics. The central hypothesis that drives this researchis that the architecture of the S. aureus biofilm is produced by changes in metabolism, thus forming distinctfunctional microniches. The rationale for this project is that understanding the metabolic and functionalheterogeneity associated with biofilm growth will provide new insights into the biology of this pathogenicorganism leading to improved therapeutic strategies. Guided by preliminary data, this objective will beachieved by attaining two specific aims: 1) Identify transcriptional changes that critically distinguish differentmicroniches and 2) Examine the functions of differentially expressed genes in biofilm development. Underthe first aim, microniches (micro-colonies and basal layer) will be identified by tracking expression offluorescent cid and lrg reporter fusions and separated using the complementary techniques of flowcytometry and laser-capture microdissection microscopy. RNA will be extracted from the microniches andgene expression differentiated and quantified via RNAseq. In the second aim, genes found to bedifferentially expressed and genes regulated by the LytSR two-component system will be tested for theireffects on micro-colony formation in a microfluidic system (BioFluxÂ®). This contribution will be significantbecause it is expected to have a significant impact on our understanding of S. aureus biofilm maturation.The proposed research is innovative because it represents a departure from the status quo, which averagesgene expression from the entire biofilm, by providing a comprehensive transcriptomic microniche-specificanalysis of the S. aureus biofilm and validation of genes identified as specific to a microenvironment using amicrofluidic device (the BiofluxÂ®) and subsequent interrogation using the Nebraska Transposon MutantLibrary. The expected outcome of the work described in this proposal is an enhanced understanding of thedevelopment of biofilms produced by S. aureus, and the metabolic diversity inherent to these structures.