Report: Condition-Specific Respiratory Chain Wiring in ADP1

Key Findings

1. Each carbon source uses a distinct respiratory chain configuration

ADP1’s branched respiratory chain (62 genes across 8 subsystems) is wired in a condition-dependent manner. Quinate requires only Complex I; acetate requires Complex I, cytochrome bo3, ACIAD3522, and more; glucose requires no specific respiratory component. This is not a quantitative gradient — it’s qualitatively different respiratory configurations per substrate.

(Notebook: 01_respiratory_chain_map.ipynb)

2. ADP1 has three parallel NADH dehydrogenases with distinct condition profiles

NDH-2 (ACIAD_RS16420, KO K03885) is TnSeq-dispensable but missing from the deletion collection — it has no growth data. FBA predicts zero flux through NDH-2 on all standard carbon sources, routing all NADH through Complex I. NDH-2 is a standalone gene (not in a respiratory operon) and is core genome.

(Notebook: 02_ndh2_indirect.ipynb)

3. The quinate-Complex I paradox is resolved by NADH flux rate, not total yield

Quinate produces FEWER NADH per carbon atom (0.57) than glucose (1.50) or acetate (1.50), yet Complex I is MORE essential on quinate. The resolution: aromatic ring cleavage via the β-ketoadipate pathway produces succinyl-CoA + acetyl-CoA simultaneously, creating a concentrated NADH burst in the TCA cycle that exceeds NDH-2’s reoxidation capacity. Glucose distributes NADH production across Entner-Doudoroff pathway steps plus TCA, staying within NDH-2’s capacity.

(Notebook: 03_stoichiometry.ipynb)

4. Cross-species: NDH-2 presence does NOT predict reduced Complex I aromatic dependency

After correcting for likely false positives in NDH-2 identification (filtering to organisms with 1-2 NDH-2 hits, excluding those with >2 hits that likely represent misannotated Complex I subunits), 5 of 14 organisms have validated NDH-2. Organisms WITH NDH-2 show LARGER Complex I aromatic deficits (mean = -0.297) than those WITHOUT (mean = -0.156), the opposite of the compensation prediction (p = 0.52, not significant). The NDH-2 compensation hypothesis is not supported by cross-species fitness data — NDH-2 presence does not predict whether Complex I is dispensable on aromatics. The ADP1 respiratory wiring pattern may be species-specific rather than a general rule.

(Notebook: 04_cross_species_respiratory.ipynb)

5. Proteomics: respiratory wiring is metabolic, not transcriptional

All three NADH dehydrogenases are expressed at similar protein levels under standard growth conditions: Complex I mean 27.6 (66th percentile), NDH-2 27.0 (59th percentile), ACIAD3522 26.2 (48th percentile), genome median 26.4. The spread is only 1.4 units. NDH-2 is NOT repressed — it is constitutively co-expressed with Complex I. This means the condition-specific respiratory wiring operates at the metabolic level: all three dehydrogenases are present simultaneously, and which one becomes limiting depends on the NADH flux rate from the carbon source being catabolized. The cell uses a passive, flux-based wiring system rather than an active transcriptional switch.

(Notebook: 05_proteomics_expression.ipynb)

Results

Respiratory Chain Inventory

ADP1 has 62 respiratory chain genes across 8 subsystems: Complex I (13 genes), NDH-2 (1), NADH-flavin oxidoreductases (5), cytochrome bo3 (4), cytochrome bd (5), Complex II/SDH (5), ATP synthase (9), and other respiratory components (20). Of these, 36 have growth data and 26 do not (including NDH-2 and several ATP synthase subunits).

The Wiring Model

The condition-specific wiring follows a biochemical logic:

• Quinate: aromatic ring cleavage → TCA burst → Complex I bottleneck. Cyt bo3 and cyt bd are dispensable because the terminal oxidase step is not the bottleneck — NADH reoxidation is.
• Acetate: direct TCA entry → continuous NADH production requiring all available dehydrogenases + cyt bo3 as the primary terminal oxidase. ACIAD3522 is specifically lethal (0.013 growth ratio).
• Lactate: pyruvate entry → moderate NADH flux + specific cyt bo3 requirement. Complex I is only mildly important (0.77), suggesting NDH-2 partially compensates.
• Glucose: Entner-Doudoroff → distributed NADH across multiple steps → NDH-2 capacity sufficient → no specific respiratory component required.

FBA Model Limitations

The FBA model predicts zero flux through NDH-2 and ACIAD3522 on all standard media, because FBA optimizes for growth rate and preferentially routes NADH through Complex I (which generates more ATP per NADH). This is the fundamental reason FBA models fail to predict condition-specific respiratory requirements — they assume the optimal pathway is always used, missing the capacity constraints that force cells to use alternative, suboptimal pathways under high flux.

Interpretation

Literature Context

• The branched respiratory chain architecture is consistent with Melo & Teixeira (2016), who described bacterial respiratory supercomplexes with multiple entry and exit points for electrons. Our data shows this branching is not just structural redundancy — it’s functionally specialized by substrate.

• NDH-2 as a low-capacity backup for Complex I is consistent with Lencina et al. (2018), who showed NDH-2 can be the sole NADH dehydrogenase in some organisms (S. agalactiae). ADP1 has both, with Complex I as the high-capacity primary system.

• The P. putida aromatic fitness data aligns with published knowledge of Pseudomonas aromatic catabolism — P. putida KT2440 is a well-studied aromatic degrader that channels diverse aromatic compounds through the β-ketoadipate pathway.

Novel Contribution

1. Condition-specific respiratory wiring map: First systematic demonstration that a single bacterium uses qualitatively different respiratory configurations for different carbon sources, not just quantitative flux adjustments.

2. Rate vs yield resolution: The paradox that quinate (lower NADH yield) requires Complex I more than glucose (higher NADH yield) is resolved by NADH flux rate — concentrated TCA burst vs distributed production. This principle should apply to any substrate whose catabolism produces concentrated reducing equivalents.

3. Three-dehydrogenase system: ADP1’s three NADH dehydrogenases (Complex I, NDH-2, ACIAD3522) with non-overlapping condition requirements demonstrate metabolic division of labor in electron transport.

4. Cross-species NDH-2 compensation NOT supported: After correcting NDH-2 false positives, organisms with validated NDH-2 show LARGER Complex I aromatic deficits than those without. The compensation is likely ADP1-specific, not a general cross-species pattern.

Limitations

• NDH-2 has no growth data — the central prediction (NDH-2 compensates on glucose) cannot be directly tested with this dataset
• The stoichiometry analysis uses theoretical pathway biochemistry, not measured flux distributions
• Cross-species comparison has only 4 organisms without NDH-2, insufficient for statistical significance
• The NDH-2 gene search in FB organisms may miss some orthologs due to variable annotation
• ACIAD3522 may not be a true NADH dehydrogenase in the respiratory sense — “NADH-FMN oxidoreductase” could serve other metabolic functions
• The NDH-2 identification in NB04 uses text matching on gene descriptions, which likely produces false positives for organisms with incompletely annotated Complex I subunits (e.g., pseudo3_N2E3 with 8 “NDH-2” hits that are probably a Complex I operon). KO-based identification (K03885) would be more reliable.
• The planned pangenome KO co-occurrence analysis (RESEARCH_PLAN Aim 2) was not performed; NDH-2/Complex I co-occurrence across Acinetobacter species remains untested. This is listed as Future Direction #3.

Data

Sources

Generated Data

Supporting Evidence

Notebooks

Figures

Future Directions

1. Construct an NDH-2 deletion mutant: The central prediction — that NDH-2 deletion makes Complex I essential on glucose — can only be tested experimentally. An NDH-2 knockout in ADP1 would be the definitive test.

2. Measure NADH/NAD⁺ ratios per carbon source: The flux rate hypothesis predicts that quinate-grown cells have higher NADH/NAD⁺ ratios than glucose-grown cells. Metabolomics could test this directly.

3. Expand cross-species analysis: With only 4 organisms lacking NDH-2, the compensation test is underpowered. Querying the full BERDL pangenome for NDH-2 (K03885) and Complex I (K00330-K00343) co-occurrence across 27K species would provide a much larger sample.

4. Characterize ACIAD3522: Is it a true respiratory NADH dehydrogenase or a metabolic enzyme? Its acetate-lethality and FMN cofactor suggest a specific role in acetate catabolism that deserves further investigation.

5. Respiratory chain proteomics: Does ADP1 upregulate Complex I on aromatic substrates and NDH-2 on glucose? Stuani et al. (2014) did quinate vs succinate proteomics — reanalyzing their data for respiratory chain proteins could test the wiring model.

References

• de Berardinis V et al. (2008). “A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1.” Molecular Systems Biology 4:174. PMID: 18319726
• Lencina AM et al. (2018). “Type 2 NADH dehydrogenase is the only point of entry for electrons into the Streptococcus agalactiae respiratory chain.” mBio 9(4):e01034-18.
• Melo AMP & Teixeira M (2016). “Supramolecular organization of bacterial aerobic respiratory chains.” BBA - Bioenergetics 1857(3):190-197.
• Price MN et al. (2018). “Mutant phenotypes for thousands of bacterial genes of unknown function.” Nature 557:503-509.
• Sena FV et al. (2024). “The two alternative NADH:quinone oxidoreductases from Staphylococcus aureus.” Microbiology Spectrum 12(4):e04152-23.
• Stuani L et al. (2014). “Novel metabolic features in Acinetobacter baylyi ADP1 revealed by a multiomics approach.” Metabolomics 10(6):1223-1238. PMID: 25374488