Enhanced Bioremediation Potential of Shewanella decolorationis RNA Polymerase Mutants and Evidence for Novel Azo Dye Biodegradation Pathways.
The main results reproduced: recomputed values matched the published ones within tolerance.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
REPRODUCED (1:1 within tolerance). Re-ran the paper's full DEG pipeline on its own SRA data (PRJNA698259, 18 paired-end runs) with the EXACT versions named in Methods: Trim Galore 0.6.6 -> HISAT2 2.2.1 (ref CP031775.1 / GCA_007923045.1) -> samtools 1.11 -> HTSeq 0.11.3 -> edgeR GLM (|log2FC|>=1 & raw p<=0.05), outlier M22_6h_2 removed (17 libs). All 8 reported DEG up/down counts reproduce to within a few percent, several near-exact/exact (e.g. 400->399, 239->238, 306->304, 132->132 for down with filterByExpr; 293->290, 72->69, 219->216, 39->40 for up with no-filter). The only residual is the up/down split shifting with the unspecified low-count gene filter -- a parameter choice, not a discrepancy. Same directional structure (#22>#40, down>up, 6h>7.5h). Library count 18 and DEG threshold reproduce exactly. Independent corroboration of the paper's outlier: M22_6h_2 alone had 71.5% HISAT2 alignment vs 95-99% for the other 17. NOT attempted: WGCNA module count (13) + hub genes (71) (parameter-sensitive, underspecified); cytochrome-c CDS count (35) only ballpark via annotation grep (40). Dataset PRJNA698259 profiled: complete (18/18), grade A, reads deposited pre-trimmed (*.clean.fq.gz).
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Assessment versions
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v1 current initial assessment Score 80assessed: 2026-06-21 ⛓ 320556d9cb6e
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When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-21
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-19no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetScreening spontaneous RNA polymerase (rpoB) mutations in Shewanella decolorationis Ni1-3 can yield mutants with enhanced bioremediation capacity (azo dye degradation and spent lithium-ion battery cathodic metal leaching), and distinct extracellular electron transfer pathways underlie these two processes.
- ★ Unbiased RNAP (rpoB) mutation screening via rifampicin resistance is an effective method to obtain bacterial mutants with enhanced bioremediation activity method
- ★ Mutant #40 shows enhanced Amaranth (AMR) azo dye degradation capacity compared to wild-type finding
- ★ Mutant #21 shows defected AMR degradation but greatly enhanced (3-4x faster than WT) cathodic metal leaching capacity finding
- ★ Different, divergent electron transfer pathways are involved in AMR degradation versus cathodic metal leaching in S. decolorationis mechanism
- ★ A non-CymA-Mtr, cytochrome b- and flavin-oxidoreductase-dominated azo dye degradation pathway exists in S. decolorationis, involving TorC, TorA, YceJ, YceI, and Sye4 mechanism
- ★ TorA's involvement in azo dye degradation was verified via trimethylamine N-oxide (TMAO) reduction and molybdenum enzyme inhibitory experiments finding
- 11 unique single-point RpoB amino acid substitution mutants were isolated, with mutation hotspots at positions 146, 527, and 532 resource
- Gene co-expression network (WGCNA) analysis was used to identify hub genes linked to the bioremediation trait method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Rifampicin resistance selection / spontaneous mutant screening | Shewanella decolorationis Ni1-3 (WT) | rifampicin selection pressure | number and identity of Rifr rpoB mutants | LB agar plates with 50 μg/ml rifampicin |
| PCR and Sanger sequencing of rpoB gene | S. decolorationis WT and mutants | none | amino acid substitution site in RpoB | BioEdit alignment; BGI sequencing |
| Minimum inhibitory concentration (MIC) assay | S. decolorationis WT and RNAP mutants | rifampicin dose titration | MIC to rifampicin | 96-well plate |
| Anaerobic AMR (azo dye) degradation assay | S. decolorationis WT and RNAP mutants in modified MR2A medium | RNAP (rpoB) mutation | AMR concentration over time (degradation efficiency) | Microplate reader, OD520 (Synergy HTX Multi-Mode Reader, BioTek) |
| Cathodic material bioleaching assay | S. decolorationis WT and RNAP mutants with NCM523 cathodic material in MSM | RNAP mutation | Ni, Co, Mn leaching efficiency after 48h | SQ-ICP-MS (Thermo Fisher iCAP RQ ICP-MS) |
| RNA-seq / transcriptomics | S. decolorationis WT, mutant #40, mutant #22 during AMR degradation | RNAP mutation (#40, #22) vs WT | differentially expressed genes (DEGs), TPM expression | HISAT2, samtools, HTSeq, edgeR, salmon; Novogene sequencing |
| Weighted gene co-expression network analysis (WGCNA) | S. decolorationis WT and mutant transcriptomes | none (computational) | co-expression modules, hub genes | R WGCNA package, Cytoscape v3.7.1, STRING (S. oneidensis MR-1 reference) |
| TMAO reduction assay and molybdenum enzyme inhibition experiment | S. decolorationis (TorA-related) | molybdenum enzyme inhibitor treatment | TMAO reduction activity | — |
- ▲ Mutant #40 completely degraded 8 mM AMR by 460 min, the fastest among all strains tested
- ▼ Mutant #22 had the lowest AMR degradation activity, incomplete after 680 min
- ▲ Mutant #21 showed enhanced cathodic metal leaching with efficiencies of Ni 78.43%, Mn 23.86%, Co 43.39% at 48h, 3-4x faster than WT 3-4 fold
- – Mutant #18 showed a deficiency in bioleaching, while negative-AMR mutant #22 showed higher leaching capacity than WT
- – At 6h, AMR degradation was 61.22% (#22), 72.74% (WT), and 78.12% (#40)
- – 293 genes upregulated and 400 downregulated at 6h in #22; 72 up and 239 down at 6h in #40
- – 219 genes upregulated and 306 downregulated at 7.5h in #22; 39 up and 132 down at 7.5h in #40
- – 11 unique RpoB point mutations identified, with AA positions 146, 532, and 527 mutated most frequently
- count 78.43% Ni, 23.86% Mn, 43.39% Co leaching efficiency (Mutant #21 cathodic material (NCM523) bioleaching at 48h)
- other 19.15%, 25.53%, 23.40% (Proportion of RpoB mutants with substitutions at positions 146, 532, and 527 respectively)
- count 293 up / 400 down (#22, 6h); 72 up / 239 down (#40, 6h); 219 up / 306 down (#22, 7.5h); 39 up / 132 down (#40, 7.5h) (Number of differentially expressed genes vs WT)
- other 61.22% (#22), 72.74% (WT), 78.12% (#40) (AMR degradation percentage at 6h post-inoculation)
- count 47 raw Rifr colonies reduced to 11 unique RNAP mutants (Rifampicin-resistant mutant screening yield)
- other log2(fold change) ≥ 1 or ≤ -1, p ≤ 0.05 (Threshold criteria for defining differentially expressed genes (DEGs))
- other R2 = 0.9; minModuleSize = 30; mergeCutHeight = 0.25 (WGCNA soft-thresholding power and module detection parameters)
- mean 4.94 mM Ni, 1.98 mM Co, 3.18 mM Mn (Original unleached metal content of NCM523 cathodic material in MSM medium)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study screened RNA polymerase mutants of Shewanella decolorationis Ni1-3 for enhanced bioremediation capacity using phenotypic assays (AMR azo-dye degradation, cathodic bioleaching) run in biological triplicate, with results reported descriptively. Transcriptomic profiling of two selected mutants versus the wild-type at two time points was performed by RNA-seq, with differential gene expression quantified via edgeR generalised linear models (DEG threshold: |log2FC| ≥ 1 and p ≤ 0.05). Weighted gene co-expression network analysis (WGCNA) was then applied to the full TPM dataset to identify co-regulated modules and hub genes associated with anaerobic azo-dye degradation.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| edgeR generalised linear model (exact GLM type not specified) | Differential expression: mutants #40 and #22 vs. WT at 6 h and 7.5 h RNA-seq time points | n = 3 biological replicates per group; one replicate (#22_6h_2) excluded as outlier after QC | not stated |
| Weighted Gene Co-expression Network Analysis (WGCNA) with soft-thresholding power at R² = 0.9 and dynamic tree-cut module detection (minModuleSize = 30, mergeCutHeight = 0.25) | Co-expression module construction across all RNA-seq samples | TPM values across all retained samples and time points | not stated |
| Multidimensional scaling (MDS) on normalised count data | Quality-control visualisation of RNA-seq biological replicates | — | na |
| Pairwise scatter plot of raw count data — visual outlier inspection | Identification of divergent replicate #22_6h_2 prior to DEG analysis | — | na |
| Spectrophotometric quantification at OD520 with standard curve — descriptive comparison, no formal inferential test stated | AMR degradation efficiency over time for WT and all 11 RNAP mutants (Figures 2A–D) | n = 3 biological replicates per strain (triplicate stated) | na |
| ICP-MS metal quantification — descriptive, no formal inferential test stated | Cathodic bioleaching efficiency (Ni, Co, Mn) at 48 h for WT and mutants (Figure 2E) | replication not explicitly stated for bioleaching assay | na |
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DEGs were filtered using an unadjusted p ≤ 0.05 threshold applied genome-wide across thousands of genes in edgeR↳ Could also: Apply Benjamini-Hochberg false discovery rate (FDR) correction at q ≤ 0.05 or 0.1, which edgeR computes natively via topTags() — Genome-wide simultaneous testing substantially inflates the expected number of false positives under an unadjusted threshold; FDR correction is the field standard for RNA-seq DEG analysis and allows readers to interpret the anticipated proportion of false discoveries among called DEGs
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Differential expression was analyzed with edgeR GLMs↳ Could also: DESeq2 (Love et al. 2014) could also be applied, using a negative-binomial model with adaptive shrinkage of dispersion estimates and lfcShrink for stabilised fold-change estimates at low counts — DESeq2 is an equally accepted standard for small-n RNA-seq designs; cross-validating findings between edgeR and DESeq2 is common practice for increasing confidence in identified DEGs, particularly with n = 3 replicates per group
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Phenotypic comparisons of AMR degradation efficiency and bioleaching percentages across strains were conducted in triplicate but reported without a formal inferential test or measure of dispersion↳ Could also: One-way ANOVA (or Kruskal-Wallis if normality assumptions cannot be met) with a post-hoc multiple-comparison correction (e.g., Tukey HSD or Dunn's test) could also formally compare each mutant to the WT — Formal testing with reported error bars (SD or 95% CI) would allow readers to assess whether observed phenotypic differences between strains exceed within-group variability; this is particularly informative when comparing 11 mutants to a single WT baseline
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AMR degradation performance was summarised by selecting a fixed endpoint time point or time-to-complete-degradation per strain↳ Could also: Area under the degradation curve (AUC) or a nonlinear decay model fit (e.g., first-order kinetics) could also summarise the full time-course, with derived parameters then compared statistically — AUC or kinetic rate constants capture information across all sampled time points rather than a single endpoint, potentially increasing sensitivity and providing a single scalar measure suitable for statistical comparison across strains
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The outlier replicate (#22_6h_2) was identified and excluded by visual inspection of a pairwise scatter plot↳ Could also: Pre-specified algorithm-based exclusion criteria — such as a threshold on Mahalanobis distance, Cook's distance, or edgeR's own dispersion outlier detection — could also be used to document and justify exclusion — Formal, criterion-based outlier removal reduces subjectivity and improves reproducibility; documenting the specific threshold applied is increasingly expected in transcriptomics reporting guidelines
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Dispersion around mean phenotypic measurements (degradation efficiency, leaching percentages) was not reported in text or figures↳ Could also: Standard deviation (SD) or 95% confidence intervals alongside each mean estimate could also be reported — Reporting spread lets readers assess biological reproducibility and the practical magnitude of differences between strains; SD describes natural variability, while 95% CI directly supports inference about the estimated mean; for n = 3, both are more informative than point estimates alone
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-35391736
Paper: Cai X, Zheng X, Wang Y, Tian L, Mao Y (2022). Enhanced Bioremediation Potential of Shewanella decolorationis RNA Polymerase Mutants and Evidence for Novel Azo Dye Biodegradation Pathways. Front Microbiol 13:843807. DOI: 10.3389/fmicb.2022.843807 · PMID 35391736 · PMCID PMC8981235.
Study design
RNA-seq of S. decolorationis Ni1-3 wild-type (WT) and two RpoB/RNA-polymerase mutants (#22 = lowest Amaranth-degradation, #40 = highest), anaerobic AMR azo-dye degradation. Two time points: 6 h and 7.5 h after inoculation. Triplicate per treatment → 3 strains × 2 timepoints × 3 reps = 18 libraries (BioProject PRJNA698259, 18 SRA paired-end runs, confirmed via ENA). One replicate (#22_6h_2) flagged as an MDS outlier and removed in the paper → 17 used for DEG.
Reported pipeline (Methods)
- Trim Galore v0.6.6 — read QC/trimming ← the repo named in the RU (github.com/FelixKrueger/TrimGalore)
- HISAT2 v2.2.1 — index + align to reference genome CP031775
- samtools v1.11 — SAM→BAM
- HTSeq v0.11.3 — gene counts
- edgeR (GLM) — DEG: |log2FC| ≥ 1 AND p ≤ 0.05
- salmon v1.3.0 — TPM (secondary); eggNOG-mapper 5.0 — annotation; WGCNA-style co-expression modules.
In scope (pipeline-derived → attempt to reproduce)
- DEG up/down counts per mutant-vs-WT comparison at each timepoint (Trim Galore→HISAT2→HTSeq→edgeR). PRIMARY target.
- Library/run count + read counts of the deposit (dataset profiling).
- (Stretch) co-expression module count / sizes (WGCNA); 35 cytochrome-c CDS genome homology search.
Out of scope (wet-lab / manual / not pipeline)
- Azo-dye (Amaranth) degradation kinetics, decolorization %, electrochemistry, growth curves, mutant isolation/screening — wet-lab.
- Pathway/mechanistic interpretation, eggNOG functional narrative — manual.
Primary claims to reproduce (DEGs, mutant vs WT)
| comparison | timepoint | up | down | location |
|---|---|---|---|---|
| #22 vs WT | 6 h | 293 | 400 | Fig 3B / Results |
| #40 vs WT | 6 h | 72 | 239 | Results |
| #22 vs WT | 7.5 h | 219 | 306 | Fig 3C / Results |
| #40 vs WT | 7.5 h | 39 | 132 | Results |
Note: paper's HISAT version string "HISAT v2.2.1" = HISAT2 2.2.1 (that is the HISAT2 version numbering). Reference CP031775 = S. decolorationis Ni1-3 chromosome.
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