PMID- 38197635 OWN - NLM STAT- MEDLINE DCOM- 20240223 LR - 20240308 IS - 1098-5530 (Electronic) IS - 0021-9193 (Print) IS - 0021-9193 (Linking) VI - 206 IP - 2 DP - 2024 Feb 22 TI - Binding of GTP to BifA is required for the production of Pel-dependent biofilms in Pseudomonas aeruginosa. PG - e0033123 LID - 10.1128/jb.00331-23 [doi] LID - e00331-23 AB - 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.IMPORTANCEc-di-GMP is the most common cyclic dinucleotide used by bacteria to regulate phenotypes such as motility, biofilm formation, virulence factor production, cell cycle progression, and cell differentiation. While the identification and initial characterization of c-di-GMP metabolic enzymes are well established, our understanding of how these enzymes are regulated to provide signaling specificity remains understudied. Here we demonstrate that the inactive GGDEF domain of BifA binds GTP and regulates the adjacent phosphodiesterase EAL domain, ultimately downregulating Pel-dependent P. aeruginosa biofilm formation through an interaction with PelD. This discovery adds to the growing body of literature regarding how hybrid GGDEF-EAL enzymes are regulated and provides additional precedence for studying how direct interactions between c-di-GMP metabolic enzymes and effectors result in signaling specificity. FAU - Van Loon, Jaime C AU - Van Loon JC AUID- ORCID: 0000-0001-8714-2922 AD - Program in Molecular Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada. AD - Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada. FAU - Whitfield, Gregory B AU - Whitfield GB AD - Program in Molecular Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada. AD - Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada. FAU - Wong, Nicole AU - Wong N AD - Program in Molecular Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada. AD - Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada. FAU - O'Neal, Lindsey AU - O'Neal L AD - Department of Microbiology, University of Washington, Seattle, Washington, USA. FAU - Henrickson, Amy AU - Henrickson A AD - Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada. FAU - Demeler, Borries AU - Demeler B AD - Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada. FAU - O'Toole, G A AU - O'Toole GA AUID- ORCID: 0000-0002-2861-4392 AD - Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA. FAU - Parsek, Matthew R AU - Parsek MR AD - Department of Microbiology, University of Washington, Seattle, Washington, USA. FAU - Howell, P Lynne AU - Howell PL AUID- ORCID: 0000-0002-2776-062X AD - Program in Molecular Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada. AD - Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada. LA - eng GR - R01 AI077628/AI/NIAID NIH HHS/United States GR - R01 AI143916/AI/NIAID NIH HHS/United States GR - R01 GM120600/GM/NIGMS NIH HHS/United States GR - R37 AI083256/AI/NIAID NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20240110 PL - United States TA - J Bacteriol JT - Journal of bacteriology JID - 2985120R RN - 0 (Bacterial Proteins) RN - 86-01-1 (Guanosine Triphosphate) RN - 0 (Escherichia coli Proteins) RN - H2D2X058MU (Cyclic GMP) RN - EC 3.1.4.- (Phosphoric Diester Hydrolases) SB - IM MH - *Pseudomonas aeruginosa/genetics/metabolism MH - Bacterial Proteins/metabolism MH - Guanosine Triphosphate/metabolism MH - *Escherichia coli Proteins/metabolism MH - Cyclic GMP/metabolism MH - Phosphoric Diester Hydrolases/metabolism MH - Biofilms MH - Gene Expression Regulation, Bacterial PMC - PMC10882990 OTO - NOTNLM OT - GTP OT - Pseudomonas OT - c-di-GMP OT - phosphodiesterase OT - regulation COIS- The authors declare no conflict of interest. EDAT- 2024/01/10 12:42 MHDA- 2024/02/23 06:43 PMCR- 2024/01/10 CRDT- 2024/01/10 09:02 PHST- 2024/02/23 06:43 [medline] PHST- 2024/01/10 12:42 [pubmed] PHST- 2024/01/10 09:02 [entrez] PHST- 2024/01/10 00:00 [pmc-release] AID - 00331-23 [pii] AID - jb.00331-23 [pii] AID - 10.1128/jb.00331-23 [doi] PST - ppublish SO - J Bacteriol. 2024 Feb 22;206(2):e0033123. doi: 10.1128/jb.00331-23. Epub 2024 Jan 10.