CRISPRi-Mediated Silencing of <i>Burkholderia</i> O-Linked Glycosylation Systems Enables the Depletion of Glycosylation Yet Results in Modest Proteome Impacts
Bibliographic record
Abstract
The process of O-linked protein glycosylation is highly conserved across the Burkholderia genus and mediated by the oligosaccharyltransferase PglL. While our understanding of Burkholderia glycoproteomes has increased in recent years, little is known about how Burkholderia species respond to modulations in glycosylation. Utilizing CRISPR interference (CRISPRi), we explored the impact of silencing of O-linked glycosylation across four species of Burkholderia; Burkholderia cenocepacia K56-2, Burkholderia diffusa MSMB375, Burkholderia multivorans ATCC17616, and Burkholderia thailandensis E264. Proteomic and glycoproteomic analyses revealed that while CRISPRi enabled inducible silencing of PglL, this did not abolish glycosylation, nor recapitulate phenotypes such as proteome changes or alterations in motility that are associated with glycosylation null strains, despite inhibition of glycosylation by nearly 90%. Importantly, this work also demonstrated that CRISPRi induction with high levels of rhamnose leads to extensive impacts on the Burkholderia proteomes, which without appropriate controls mask the impacts specifically driven by CRISPRi guides. Combined, this work revealed that while CRISPRi allows the modulation of O-linked glycosylation with reductions up to 90% at a phenotypic and proteome levels, Burkholderia appears to demonstrate a robust tolerance to fluctuations in glycosylation capacity.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.005 | 0.003 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 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 teacher head, 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".