The Pseudomonas fluorescens Pho regulon and its role in modulating biofilm formation in response to environmental cues

Microbial biofilm formation is thought of as a developmental pathway, whereby cells progress through environmentally regulated and temporally distinct stages during transition from free-swimming lifestyles to members of a structured surface-attached consortium. The focus of my doctoral studies has been to understand how environmental cues are sensed by P. fluorescens and integrated with mechanisms to regulate an important step in biofilm formation, the transition to committed association with the surface.;Low levels of extracellular inorganic phosphate (Pi) were shown to inhibit stable surface attachment by P. fluorescens. Activation of the Pho regulon in Pi-limiting conditions was shown to be both necessary and sufficient for inhibition of biofilm formation. The protein product of the Pho regulon gene rapA cleaves the intracellular signaling molecule c-di-GMP. Reductions in the levels of c-di-GMP were shown to inhibit both secretion and localization of LapA to the outer membrane. LapA is a large adhesin that is absolutely required for P. fluorescens to form stable attachments with surfaces.;Studies were also performed to identify pathways independent of P i signaling that regulate Pho activation. Mutations to Pfl5137 were found to inhibit Pho regulon activation and promote biofilm formation by P. fluorescens. Pfl5137 was identified as a diadenosine tetraphosphatase homologous to ApaH from E. coli. Loss of Pfl5137 resulted in elevated levels of the nucleotide derivative diadenosine tetraphosphate (Ap4A), which is thought to be an intracellular signal involved in adaptation to oxidative stress. In P. fluorescens, elevated levels of Ap4A correlated with increases in lapA transcription and secretion of LapA to the cell surface. Similar to c-di-GMP, it is possible that Ap4A may serve to connect extracellular signals to regulation of surface attachment via regulation of Lap system function.;Overall, this study provides strong evidence for the existence of specific molecular mechanisms that couple the extracellular environment to regulation of a specific stage in biofilm formation, the commitment of a cell to life on a surface.