Pseudomonas fluorescens is a soil dwelling organism with biological control activity.  That is, this organism can protect certain plants from pathogens in the soil. P. fluorescens likely occupies 3 key niches in the environment. 

Recent work in the O’Toole lab has focused on two aspects of the transition of P. fluorescens from a planktonic to a surface-attached lifestyle:  Integration of environmental signals that modulate biofilm formation (i.e. regulation) and structural genes required for surface attachment.

The ‘core’ biofilm machinery:

Our laboratory identified a group of genes absolutely required for biofilm formation by P. fluorescens on abiotic surfaces.  This locus, designated lap for "large adhesin protein", encodes a >500 kDa protein, LapA, and an ABC transporter required for its secretion.  LapA is an adhesin that associates with the surface of cells, and is required for their transition from reversible to irreversible attachment to a surface (1).  Since our description of the lap locus, similar adhesins have been identified in other bacteria, suggesting that LapA is a member of a conserved family adhesins. 

Below is a diagram of the lap locus and gene products, including their localization in the bacterial cell.  Mutations in lap transporter genes block secretion of LapA from the cell cytoplasm to the cell surface.

We have also identified a fifth gene at the lap locus that is required for surface attachment under most conditions.  Mutants for this gene, lapD, fail to localize the LapA adhesin to the cell surface and, like lapA mutants, show a defect in the transition to irreversible surface attachment (2).  The LapD protein localizes to the inner membrane of P. fluorescens, and contains predicted Di-guanylate cyclase (DGC) and cyclic di-GMP phosphodiesterase (PDE) domains.  Work in our lab, and others, has demonstrated an important role for c-di-GMP signaling in biofilm formation by Pseudomonads (see below).  Ongoing research aims to identify LapD’s role in c-di-GMP signaling, and the mechanism by which it effects adhesin localization. 

Environmental signals and regulation of biofilm formation:

Inorganic phosphate is an essential nutrient that is typically found associated with surfaces in soil environments – soluble phosphate is typically found at concentrations below 2 mM.  We have shown that P. fluorescens responds to phosphate limitation by induction of the Pho regulon (genes regulated by the PhoBR two component system)(3).  In addition to the up-regulation of genes involved in phosphate scavenging, Pho induction blocks biofilm formation through production of a c-di-GMP phosphodiesterase, RapA.  c-di-GMP is an intracellular second-messenger that has been shown to regulate surface-associated behaviors in pseudomonads and other bacteria.  During phosphate starvation, RapA expression leads to a decrease in cellular levels of c-di-GMP.  Decreased c-di-GMP levels, in turn, block the secretion and surface localization of LapA, the adhesin required for attachment to surfaces (4).  This represents a novel role for the second messenger, and we are currently working to identify the mechanism by which c-di-GMP regulates adhesin transport and localization. 

A current working model:

Above is a general diagram of our current model that integrates the regulation of biofilm by phosphate with the roles of the lap proteins (dotted arrows represent topics under investigation).

Low levels of environmental phosphate are sensed by PhoR, which activates PhoB through phosphorylation.  Phosphorylated PhoB activates the transcription of RapA (as well as other members of the pho regulon).  RapA cleaves c-di-GMP, depleting cellular pools of this signaling molecule.  Both c-di-GMP levels and LapD have effects on the secretion and localization of the LapA adhesin.

Our recent work has focused on how the cdiGMP signal, whose level is controlled in part by the RapA enzyme, controls the localization of the LapA adhesion on the cell surface.  A recent report from our group has shown that LapD is a novel cdiGMP binding protein, and furthermore, binding of cdiGMP by LapD transmits a signal to the periplasm.  Thus, our current model is that LapD, via a direct or indirect mechanism, mediates LapA localization via Pi-dependent changes in the cdiGMP pool.  Our current overall model is shown below.

Relevant Publication(s):

1.  Hinsa SM, Espinosa-Urgel M, Ramos JL, O'Toole GA. Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein.  Mol Microbiol. 2003 Aug;49(4):905-18. 

2.  Hinsa SM and O’Toole GA.  Biofilm formation by Pseudomonas fluorescens WCS365: a role for LapD.  Microbiology. 2006; 152: 1375-1383. 

3.  Monds RD, Newell PD, Schwartzman JA, O’Toole GA.  Conservation of the Pho regulon in Pseudomonas fluorescens Pf0-1.  Appl. Environ. Microbiol.  2006; 72, 3: 1910-1924. 

4.  Monds RD, Newell PD, Gross RH, O’Toole GA.  Phosphate-dependent modulation of c-di-GMP levels regulates Pseudomonas fluorescens Pf0-1 biofilm formation by controlling secretion of the adhesin LapA.  Mol. Microbiol. 2007; 63,3: 656-679.

5. Newell PD, Monds RD, O'Toole GA. 2009. LapD is a bis-(3',5')-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0-1.  Proc Natl Acad Sci U S A. 106:3461-6.