LapD is a bis-(3',5')-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0-1

Abstract

The second messenger cyclic dimeric GMP (c-di-GMP) regulates surface attachment and biofilm formation by many bacteria. For Pseudomonas fluorescens Pf0-1, c-di-GMP impacts the secretion and localization of the adhesin LapA, which is absolutely required for stable surface attachment and biofilm formation by this bacterium. In this study we characterize LapD, a unique c-di-GMP effector protein that controls biofilm formation by communicating intracellular c-di-GMP levels to the membrane-localized attachment machinery via its periplasmic domain. LapD contains degenerate and enzymatically inactive diguanylate cyclase and c-di-GMP phosphodiesterase (EAL) domains and binds to c-di-GMP through a degenerate EAL domain. We present evidence that LapD utilizes an inside-out signaling mechanism: binding c-di-GMP in the cytoplasm and communicating this signal to the periplasm via its periplasmic domain. Furthermore, we show that LapD serves as the c-di-GMP receptor connecting environmental modulation of intracellular c-di-GMP levels by inorganic phosphate to regulation of LapA localization and thus surface commitment by P. fluorescens.

Fig. 1.

Biofilm formation and LapA localization phenotypes of the lapD mutant. (A) Quantitative analysis of biofilm formation by WT plus vector (WT pvect), ΔlapD plus vector (pvect), and ΔlapD plus pLapD (pLapD). (B) Western blots probed for LapA to analyze adhesin localization profiles for strains shown in (A). The fractions indicated are cellular (Cell), cell-associated (CA), and culture supernatant (S). (C) Predicted protein domains of LapD.

Fig. 2.

Analysis of c-di-GMP binding by LapD. (A) TLC resolution of [³²P] c-di-GMP co-purified with histidine-tagged proteins. Each reaction contained 50 μg of protein unless otherwise indicated. HD = heat denatured. (B) Increasing amounts of unlabeled c-di-GMP decrease binding of LapD to [³²P] c-di-GMP (Left), whereas unlabeled GTP does not compete with [³²P] c-di-GMP binding at concentrations up to 1 mM (1000-fold excess) (Right). (C) c-di-GMP binding by LapD lacking the GGDEF or EAL domain or by the EAL domain alone. (D) Binding of c-di-GMP to E. coli membranes containing LapD (diamonds) or the ΔEAL protein (open squares) at increasing concentrations of ligand. Specific binding (dark squares) is binding to LapD minus binding to ΔEAL.

Fig. 3.

Biochemical and phenotypic analysis of EAL-domain point mutants. (A) Membrane preparations were assayed for in vivo levels of LapD by Western blot. (B) c-di-GMP binding by LapD variants and WT LapD. (C) Biofilm assay performed on ΔlapD complemented with pLapD or EAL-domain mutants. (D) Strains with the ΔlapD, WT, or K446A allele of lapD are compared for biofilm formation when the DGC PA1107 is expressed.

Fig. 4.

Effects of lapD mutations on biofilm formation and cell-surface LapA levels. (A) Mutant forms of lapD are compared with pLapD in their ability to complement ΔlapD for biofilm formation. (B) Quantification of cell-surface levels of LapA in the strains in (A) by densitometry on α-LapA dot blots (n = 3, ± SD; representative blots are shown).

Fig. 5.

The role of LapD in the phosphate-regulated c-di-GMP signaling pathway. (A) Quantitative assay of biofilm formation by the strains indicated. (B) Surface attachment by the indicated strains was monitored in high- and low-Pi media. The percentage of attachment in low-Pi medium relative to high-Pi medium is given at 0, 60, and 120 min after Pi starvation. (C) Surface attachment by the indicated strains in high-Pi medium at 0, 60, and 120 min after ectopic induction of rapA expression (given as a percentage of attachment by the isogenic strain without inducer at each time point).

Fig. 6.

A model for inside-out signaling through LapD. (Left) In the absence of c-di-GMP binding, the periplasmic output is repressed via the HAMP domain. (Right) When c-di-GMP is bound by LapD, the HAMP domain assumes a conformation that activates output. LapD is depicted as a dimer, because other HAMP domain proteins are known to be dimers.