| Biofilm-related infections are extremely costly due to their increased tolerance to antibiotics they confer. Unfortunately, current therapeutic strategies are primarily based on the ability of antibiotics to kill planktonic cells. Thus, the development of effective therapies will require a basic understanding of biofilm physiology. Recent studies of various bacterial species demonstrate an important role for cell death and lysis during the formation of biofilm. The work described in this dissertation seeks to gain a better understanding of two known regulators of cell death and lysis, the cid and lrg loci in S. aureus, and their roles in bacterial programmed cell death (PCD) and biofilm development. The results of this work demonstrate that a mutation in the cid operon, encoding a putative holin-like effector of lysis, caused decreased cell lysis and release of genomic DNA (termed eDNA) during biofilm growth, leading to decreased biofilm adherence. In contrast, the lrg operon, which encodes a putative antiholin-like inhibitor of cell lysis, was shown to inhibit eDNA release, affecting biofilm formation. To test the hypothesis that the differential expression of the cid and lrg operons are important for the control of death within a subpopulation of cells, transcriptional reporter fusions were generated to assay cid and lrg transcription in individual cells. Immature S. aureus biofilm has cidABC expression in dense microcolonies while lrgAB expression at the same developmental stage is minimal. As the biofilm matures cidABC expression is observed in the basal biofilm layers and only limited expression in the tower structures. Expression of lrgAB appears on the top and outside of tower formations primarily and less in the basal biofilm areas. An ldh1 transcriptional reporter, indicating low oxygen microenvironments, correlated with the same pattern of biofilm expression as the cidABC reporter. Overall, the results demonstrate the importance of the cid and lrg operons in biofilm formation by controlling death and lysis of a subpopulation of cells and allowing for eDNA release. Furthermore, they provide the first evidence for the developmental control of these operons and support the model that they play a key role in the control of PCD during biofilm formation. |
