We study how the intracellular metabolites c-di-GMP modulates the expression and activities of genes and proteins required for biofilm formation by the opportunistic pathogen and model biofilm organism Pseudomonas. aeruginosa. Biofilms, defined as surface-associated multicellular communities encased in a self-produced extrapolysaccharide matrix, are responsible for chronic P. aeruginosa infections in humans with underlying predispositions such as cystic fibrosis. Biofilm infections are problematic because they are resistant to antibiotic treatment and tend to escape immune surveillance.

High intracellular cyclic di-GMP  (c-di-GMP) promotes biofilm formation in many bacterial species. Like other gram-negative bacteria, P. aerguinosa has many genes encoding proteins with predicted GGDEF domains responsible for the synthesis of c-di-GMP. It also has genes with predicted EAL domains for c-di-GMP hydrolysis.

The general parameters of c-di-GMP activity have been established, but environmental inputs that stimulate c-di-GMP synthesis, intracellular trafficking of c-di-GMP and mechanisms of c-di-GMP action are just beginning to be explored in bacteria.
.
We discovered that c-di-GMP binds to a transcription factor named FleQ to derepress expression of exopolysaccharide synthesis genes, as well as other genes important for biofilm formation. We continue to study the functioning of FleQ at the molecular level.

In related work we are studying the activity and subcellular localization characteristics of a GGDEF protein called WspR as an avenue to expand our knowledge of the behavior of a GGDEF protein and accompanying c-di-GMP production in individual cells. Our data suggest that growth on an agar surface stimulates the activity of a Wsp signal transduction system that in turn stimulates WspR to synthesize c-di-GMP. This supports the notion that a condition associated with cell-to-cell contact or cell-surface interactions stimulates P. aeruginosa to form biofilms. 

These studies involve protein biochemistry, mutagenesis and fluorescence microscopy.