LapG, required for modulating biofilm formation by Pseudomonas fluorescens Pf0-1, is a calcium-dependent protease.
Biofilm formation by Pseudomonas fluorescens Pf0-1 requires the cell surface adhesin LapA. We previously reported that LapG, a periplasmic cysteine protease of P. fluorescens, cleaves the N terminus of LapA, thus releasing this adhesin from the cell surface and resulting in loss of the ability to make a biofilm. The activity of LapG is regulated by the inner membrane-localized cyclic-di-GMP receptor LapD via direct protein-protein interactions. Here we present chelation and metal add-back studies demonstrating that calcium availability regulates biofilm formation by P. fluorescens Pf0-1. The determination that LapG is a calcium-dependent protease, based on in vivo and in vitro studies, explains the basis of this calcium-dependent regulation. Based on the crystal structure of LapG of Legionella pneumophila in the accompanying report by Chatterjee and colleagues (D. Chatterjee et al., J. Bacteriol. 194:4415-4425, 2012), we show that the calcium-binding residues of LapG, D134 and E136, which are near the critical C135 active-site residue, are required for LapG activity of P. fluorescens in vivo and in vitro. Furthermore, we show that mutations in D134 and E136 result in LapG proteins no longer able to interact with LapD, indicating that calcium binding results in LapG adopting a conformation competent for interaction with the protein that regulates its activity. Finally, we show that citrate, an environmentally relevant calcium chelator, can impact LapG activity and thus biofilm formation, suggesting that a physiologically relevant chelator of calcium can impact biofilm formation by this organism.
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
Chelsea D
CD
Boyd
Debashree
D
Chatterjee
Holger
H
Sondermann
George A
GA
O'Toole
D019943:Amino Acid Substitution; D001665:Binding Sites; D018441:Biofilms / Q000254:growth & development*; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000378:metabolism; D019343:Citric Acid / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011487:Protein Conformation; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D002614:Chelating Agents; D003067:Coenzymes; D050505:Mutant Proteins; D019343:Citric Acid; D003546:Cysteine Endopeptidases; D002118:Calcium