Binding of GTP to BifA is required for the production of Pel-dependent biofilms in <i>Pseudomonas aeruginosa</i>
Bibliographic record
Abstract
ABSTRACT 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. IMPORTANCE c-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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".