H-065. LapD is a Cyclic di-GMP Binding Protein Required for Localization and Stability of the LapA Adhesin in Pseudomonas fluorescens Pf0-1

P. D. Newell, G. A. O'Toole, Jr;
Dartmouth Med. Sch., Hanover, NH.

Background: LapD is an inner membrane protein required for biofilm formation by Pseudomonas fluorescens Pf0-1. It contains a predicted cyclic-di-GMP (c-di-GMP) phosphodiesterase (PDE) domain, and a predicted diguanylate cyclase domain (DGC). A mutation in lapD leads to the loss of the LapA adhesin from the outer membrane, and thus an inability to stably attach to surfaces. The mechanism by which LapD affects LapA localization is unknown. To gain further insight into LapD’s function, we characterized its biochemical activity in vitro and assessed the function of mutant LapD alleles. Methods: A Histidine-tagged LapD protein (LapD-6H) was expressed in E. coli and purified by nickel affinity chromatography. PDE and DGC activities were tested in vitro alongside published controls. Binding of LapD to c-di-GMP was assessed using two methods: (1) co-precipitation of [32]P labeled c-di-GMP with LapD-6H bound to nickel resin and (2) crosslinking of soluble c-di-GMP-LapD complexes by UV irradiation. LapD variants were generated in three ways: site directed mutagenesis of conserved amino acid residues, sequential deletion of the predicted domains of LapD, and a complementation screen of mutant alleles generated by error-prone PCR. Function of mutant alleles was tested in vitro using c-di-GMP binding assays and in vivo through complementation. Results: While the LapD protein has neither PDE nor DGC activity in vitro, LapD binds the second messenger c-di-GMP in a specific and saturable manner. Quantitative binding assays estimate the affinity (Kd) of this interaction to be 49±4.5uM. Functional analyses of purified LapD variants demonstrate that the predicted PDE domain of LapD is necessary and sufficient for c-di-GMP binding. Mutant proteins with altered binding affinity fail to complement lapD mutants. Conclusions: LapD does not metabolize c-di-GMP but does bind this molecule, and binding is required for function in vivo. Our structure/function analyses support a model in which c-di-GMP binding in the cytoplasm modulates a periplasmic output that is necessary and sufficient for LapD’s affects on LapA and biofilm formation.