Molecular and biochemical characterization of ExeAB, two components involved in the type II secretion pathway of <i>Aeromonas hydrophila</i>.
Bibliographic record
Abstract
The objective of this study was to investigate the role of ExeA in the Aeromonas hydrophila type II secretion pathway.Pulse-chase analysis indicated that ExeA was stabilized by coexpression with ExeB.From in vivo cross-linking analyses, both proteins were found to participate in a complex of approximately 85 kDa, each requiring the presence of the other protein.Radiolabeling followed by cross-linking, immunoprecipitation and then reduction of the cross-links confirmed the presence of the two proteins in the same complex.The complex could also be extracted intact from cell membranes with non-ionic detergents.In addition, gel filtration analysis performed with octyl-glucoside soluble membrane extracts suggested the existence of a native heterododecameric ExeAB complex.Examination of a collection of ExeA substitution derivatives, altered in consensus ATP binding/hydrolysis motifs located near its N-terminus, suggested that ATP binding by ExeA is required for ExeAB complex formation, while hydrolysis is required for its function in secretion.To directly examine ExeA function, the N-terminal cytoplasmic domain of ExeA with the addition of a C-terminal hexahistidine tag (cytExeA) was overproduced in Escherichia coli and purified by metal chelate affinity chromatography followed by anion-exchange chromatography.Purified preparations of cytExeA were found to exhibit ATPase activity dependent on the presence of a divalent cation.Enzymatic characterization of cytExeA revealed an optimum reaction temperature of approximately 37-40 °C and a pH of 7-8.As well, Mg2+ was the preferred cation;cytExeA exhibited an apparent Km for Mg-ATP of 1.2 mM and a V.), of 1.1 nmoles mind mg-I of protein.cytExeA was also able to utilize CTP, GTP and UTP as substrates.As well, inhibitors of F-type ATPases were found to significantly reduce cytExeA
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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) | 0.001 | 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) | 0.002 | 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".