Separation of <i>Pseudomonas aeruginosa</i> type IV pilus-dependent twitching motility and surface-sensing responses
Bibliographic record
Abstract
ABSTRACT Many functions of Pseudomonas aeruginosa type IVa pili, including twitching motility and surface sensing, depend on dynamic cycles of filament assembly and disassembly powered by the cytoplasmic ATPases PilB, PilT, and PilU. Deletion of pilT results in loss of twitching and pilus-specific bacteriophage susceptibility, while non-twitching pilU mutants remain susceptible to pilus-specific phages, indicating that they still produce retractable pili. pilU mutants have high basal levels of the secondary messenger cyclic AMP (cAMP) that normally increases following surface contact, suggesting aberrant surface sensing. To better understand PilU’s role in pilus biology, we solved its X-ray crystal structure and used phylogenetic analyses to identify conserved differences between PilT and PilU. Chemical mutagenesis followed by whole-genome sequencing was used to identify suppressors in the Δ pilU mutant background that restored twitching motility. The mutations mapped to the major pilin, PilA, or the pilus tip adhesin, PilY1. Both the position and nature of the substitutions in PilA impacted restoration of motility, and it was dependent on functional PilT. Complementation of most pilU suppressors with PilU in trans further increased motility, while the expression of wild-type PilA in trans decreased motility in a dose-dependent manner. Notably, cAMP levels remained elevated in most twitching pilU suppressor mutants, showing that surface sensing and motility can be uncoupled. Together, our data suggest that the bacterial response to surfaces reflects a complex interaction of PilU function with specific alleles of PilY1 and PilA that together modulate pilus dynamics and function. IMPORTANCE The ability of bacteria to sense and respond to contact with surfaces is important for triggering changes in secondary messenger levels and gene expression, leading to the formation of biofilms and increased production of virulence factors. For Pseudomonas aeruginosa , the expression of functional type IVa pili is important for the accumulation of cyclic AMP (cAMP) following surface contact. Deletion of the PilT retraction ATPase paralog PilU leads to loss of pilus-mediated twitching motility but also high intracellular levels of cAMP, a phenotype mimicking that of surface-adapted cells. Here, we isolated twitching suppressors of a pilU deletion mutant that mapped to the pilin subunit PilA or pilus-tip adhesin PilY1 and showed that for most, elevated cAMP levels did not decrease when motility was restored. Twitching was dependent on functional PilT, and complementation with PilU further increased twitching for most mutants. These data show that in permissive contexts, PilU is not required for twitching motility, providing new insights into mechanisms of bacterial surface sensing and evolution of type IVa pilus motor function.
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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 | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
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".