Binding of GTP to BifA is required for the production of Pel-dependent biofilms in Pseudomonas aeruginosa

Jaime C. Van Loon, Gregory B. Whitfield, Nicole Wong, Lindsey O'Neal, Amy Henrickson, Borries Demeler, G. A. O'Toole, Matthew R. Parsek, P. Lynne Howell

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


The Pel exopolysaccharide is one of the most mechanistically conserved and phylogenetically diverse bacterial biofilm matrix determinants. Pel is a major contributor to the structural integrity of Pseudomonas aeruginosa biofilms, and its biosynthesis is regulated by the binding of cyclic-3′,5′-dimeric guanosine monophosphate (c-di-GMP) to the PelD receptor. c-di-GMP is synthesized from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases with GGDEF domains and degraded by phosphodiesterases with EAL or HD-GYP domains. As the P. aeruginosa genome encodes 43 c-di-GMP metabolic enzymes, one way signaling specificity can be achieved is through direct interaction between specific enzyme-receptor pairs. Here, we show that the inner membrane hybrid GGDEF-EAL enzyme, BifA, directly interacts with PelD via its cytoplasmic HAMP, GGDEF, and EAL domains. Despite having no catalytic function, the degenerate active site motif of the BifA GGDEF domain (GGDQF) has retained the ability to bind GTP with micromolar affinity. Mutations that abolish GTP binding result in increased biofilm formation but stable global c-di-GMP levels. Our data suggest that BifA forms a dimer in solution and that GTP binding induces conformational changes in dimeric BifA that enhance the BifA-PelD interaction and stimulate its phosphodiesterase activity, thus reducing c-di-GMP levels and downregulating Pel biosynthesis. Structural comparisons between the dimeric AlphaFold2 model of BifA and the structures of other hybrid GGDEF-EAL proteins suggest that the regulation of BifA by GTP may occur through a novel mechanism.

Original languageEnglish
Pages (from-to)e0033123
JournalJournal of Bacteriology
Issue number2
StatePublished - Feb 2024


  • GTP
  • Pseudomonas
  • c-di-GMP
  • phosphodiesterase
  • regulation
  • Cyclic GMP/metabolism
  • Pseudomonas aeruginosa/genetics
  • Phosphoric Diester Hydrolases/metabolism
  • Bacterial Proteins/metabolism
  • Biofilms
  • Escherichia coli Proteins/metabolism
  • Guanosine Triphosphate/metabolism
  • Gene Expression Regulation, Bacterial


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