Biofilm Formation and Quorum Sensing in Pseudomonas fluorescens Pf0-1
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A bacterial biofilm is a community of microorganisms adhering to a surface, exhibiting biochemical and phenotypic differences from their planktonic counterparts. The transition from a free-floating to sessile cell type has been shown to be, in part, mediated by high intracellular levels of the nucleotide second messenger c-di-GMP. It is suggested that one of the environmental cues for biofilm formation, recognized by members of the c-di-GMP network, is local cell density. In areas of high cell density, cells can communicate through a system called quorum sensing. In gram negative bacteria, acyl-homoserine lactone (AHL) molecules are excreted into the surrounding medium and recognized by cells in close proximity. It is hypothesized that upon recognizing AHLs through c-di-GMP signaling, gene expression is altered leading to a sessile lifestyle. Thus, the long-term goal of this research is to provide evidence for the link between c-di-GMP and quorum sensing-mediated mechanisms in biofilm formation in Pseudomonas fluorescens Pf0-1. The first objective towards this goal was to identify the AHLs utilized by P. fluorescens Pf0-1 in quorum sensing mechanisms. Through gas-chromatography mass-spectrometry (GCMS), two AHLs were identified in the supernatant of P. fluorescens Pf0-1; N-butyryl-HSL and Ndecanoyl- HSL. Subsequent work will focus on the identification of AHLs with longer acyl-chain and varying levels of acyl chain oxidation. The second objective towards this goal was to utilize the 96-well static microtiter plate biofilm assay as a platform for studying the relationships between c-di-GMP and AHL-mediated mechanisms in biofilm formation. As a protocol for 96-well static biofilm assays that was previously successful was no longer reproducible, different microtiter plate surfaces were surveyed for their ability to support P. fluorescens Pf0-1 biofilm and to investigate potential factors that could interfere with development on the abiotic surface. Throughout the troubleshooting process, biofilm assay experiments carried out in microtiter plates with the same type of surface chemistry, but from different manufacturers and batches, resulted in variable quantities of biofilm. This observation then inspired the production of a surface that would create more favorable interactions with bacterial cells and offer increased points of attachment to further promote biofilm formation. In this new platform, microtiter plates are pre-treated by abrasive forces such as sandblasting and drilling before biofilm assays, which gives robust biofilm formation that will allow for future investigation into connections between c-di-GMP and AHL-controlled mechanisms of biofilm formation in P. fluorescens Pf0-1.