Differential Impact of MexB Mutations on Substrate Selectivity of the MexAB-OprM Multidrug Efflux Pump of <i>Pseudomonas aeruginosa</i>
Bibliographic record
Abstract
The integral inner membrane resistance-nodulation-division (RND) components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems. To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a DeltamexB strain. Of 100 mutants that expressed wild-type levels of MexB and showed increased susceptibility to one or more of carbenicillin, chloramphenicol, nalidixic acid, and novobiocin, the mexB genes of a representative 46 were sequenced, and 19 unique single mutations were identified. While the majority of mutations occurred within the large periplasmic loops between transmembrane segment 1 (TMS-1) and TMS-2 and between TMS-7 and TMS-8 of MexB, mutations were seen in the TMSs and in other periplasmic as well as cytoplasmic loops. By threading the MexB amino acid sequence through the crystal structure of the homologous RND transporter from Escherichia coli, AcrB, a three-dimensional model of a MexB trimer was obtained and the mutations were mapped to it. Unexpectedly, most mutations mapped to regions of MexB predicted to be involved in trimerization or interaction with MexA rather than to regions expected to contribute to substrate recognition. Intragenic second-site suppressor mutations that restored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction. As the G220S mutation likely impacted trimerization, it appears that either proper assembly of the MexB trimer is necessary for OprM interaction or OprM association with an unstable MexB trimer might stabilize it, thereby restoring activity.
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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 | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 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 | 0.000 |
| 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".