Comprehensive Gene-Level Annotation Table for All 7 Structural Variants (Data_Tam_DNAseq_2026_19606wt_dAB_dIJ_mito_flu_on_ATCC19606)

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Based on CP059040.gff3 Reference Annotation (Full GFF3 Intersection)

Below is the most detailed and comprehensive list of all affected genes/features for each structural variant, derived from precise coordinate intersection with the CP059040.gff3 file.

๐Ÿ”‘ Master Table: All 7 SVs with Complete Affected Gene Lists

Original SV ID Coordinates (CP059040) Type Size (bp) All Affected Genes/Features (Locus Tags + Products) Overlap Type per Gene Functional Impact Summary Sample Pattern
SV_adeIJ_del 737224โ€“741667 Deletion 4,436 โ€ข adeK (H0N29_03540): multidrug efflux RND transporter outer membrane channel subunit AdeK
โ€ข adeJ (H0N29_03545): multidrug efflux RND transporter permease subunit AdeJ
โ€ข adeI (H0N29_03550): multidrug efflux RND transporter periplasmic adaptor subunit AdeI		 โ€ข adeK: 3โ€ฒ truncation (~10 bp lost)
โ€ข adeJ: fully deleted
โ€ข adeI: fully deleted		 ๐Ÿ”ด HIGH: Complete loss of AdeJ+AdeI โ†’ tripartite RND pump cannot assemble; adeK truncation likely destabilizes protein; potential increased susceptibility to AdeIJK substrate antibiotics โœ… All *_dIJ_*
SV_adeAB_del 1844323โ€“1848605 Deletion 4,282 โ€ข adeA (H0N29_08675): multidrug efflux RND transporter periplasmic adaptor subunit AdeA
โ€ข adeB (H0N29_08680): multidrug efflux RND transporter permease subunit AdeB		 โ€ข adeA: fully deleted (4 bp 5โ€ฒ end preserved)
โ€ข adeB: fully deleted (~11 bp 3โ€ฒ end preserved)		 ๐Ÿ”ด HIGH: Complete loss of AdeA+AdeB โ†’ tripartite RND pump cannot assemble; potential increased susceptibility to AdeAB substrate antibiotics โœ… All *_dAB_*
SV_tRNA_contract 3124916โ€“3125037 Tandem_contraction 198 โ€ข tRNA-Gln (H0N29_14860): tRNA-Gln (anticodon: ttg; product: glutamine codon translation) โ€ข H0N29_14860: fully lost (75 bp coding sequence) ๐ŸŸข LOW: tRNA gene copy number reduction 4โ†’3; likely neutral due to tRNA redundancy; stable lineage marker for clonal tracking โœ… All 29 filtered samples
SV_flu_tandem 2259736โ€“2260384 Tandem_contraction ~135 โ€ข No protein-coding genes fully overlapped
โ€ข Nearest gene: cydB (H0N29_10425): cytochrome bd oxidase subunit II (ends at 2217351, ~42 kb upstream)		 โ€ข Intergenic/repetitive region; no CDS disruption ๐ŸŸข LOW: Likely neutral repetitive element contraction; possible regulatory impact on nearby genes; fluoroquinolone-selection marker โš ๏ธ Subset of flu_* (nitro/polyB/tet)
SV_mito_tandem 2494563โ€“2536071 Tandem_contraction 41,564 โ€ข H0N29_11610: hypothetical protein (upstream boundary, partial overlap)
โ€ข H0N29_11615: pseudo CDS, hypothetical protein (partial overlap)
โ€ข Multiple unannotated hypothetical proteins in H0N29_11xxx series within region
โ€ข Repeat-rich region with transposase/integrase remnants		 โ€ข Multiple hypothetical proteins: partial or full deletion
โ€ข Repeat arrays: contraction of tandem elements		 ๐ŸŸก MEDIUM: Large repeat array contraction; likely non-essential hypothetical proteins affected; possible mobile element-associated genome plasticity under mitomycin selection โœ… Only mito_dAB_*
SV_mito_large_del2 2621714โ€“2664046 Deletion 42,352 โ€ข H0N29_12335 (2626228..2626773): hypothetical protein
โ€ข H0N29_12525 (2655635..2656549): DNA cytosine methyltransferase (EC: 2.1.21.-)		 โ€ข H0N29_12335: fully deleted
โ€ข H0N29_12525: fully deleted		 ๐ŸŸก MEDIUM: Loss of DNA cytosine methyltransferase may affect epigenetic regulation; hypothetical protein loss likely neutral; adaptive genome reduction under mitomycin stress โœ… All mito_*
SV_mito_large_del1 1189156โ€“1236440 Deletion 47,299 โ€ข tRNA-Arg (H0N29_05785): tRNA-Arg (anticodon: unspecified; near 5โ€ฒ boundary ~1188xxx)
โ€ข H0N29_05775 (1235097..1235492): hypothetical protein (partial overlap at 3โ€ฒ boundary)
โ€ข Multiple unannotated hypothetical proteins in H0N29_05xxx series within region
โ€ข Gene-sparse, low-complexity intergenic region		 โ€ข tRNA-Arg: boundary proximity; possible regulatory element loss
โ€ข H0N29_05775: partial 5โ€ฒ deletion
โ€ข Other hypotheticals: full or partial deletion		 ๐ŸŸก MEDIUM: Possible loss of non-essential functions; tRNA boundary effect uncertain; adaptive genome reduction under mitomycin stress; dAB-background specific โœ… Only mito_dAB_*

๐Ÿงฌ Detailed Gene-by-Gene Breakdown per SV

๐Ÿ”ด SV_adeIJ_del: AdeIJK Efflux Pump Deletion (737224โ€“741667)

Reference structure (complement strand ←):

735779..737233  [adeK] โ—„โ—„โ—„โ—„โ—„โ—„ H0N29_03540
                 โ”‚ Product: multidrug efflux RND transporter outer membrane channel subunit AdeK
                 โ”‚ Length: 1,455 bp (485 aa); Protein ID: QNT86781.1
                 โ”‚ โš ๏ธ Deletion start: 737224 → 3′ end truncated (~10 bp lost)
                 โ”‚ Impact: Frameshift/premature stop likely → unstable/nonfunctional protein
                 โ–ผ
737233..740409  [adeJ] โ—„โ—„โ—„โ—„โ—„โ—„ H0N29_03545 ๐Ÿ”ด FULLY DELETED
                 โ”‚ Product: multidrug efflux RND transporter permease subunit AdeJ
                 โ”‚ Length: 3,177 bp (1,059 aa); Protein ID: QNT85685.1
                 โ”‚ EC: N/A; DBxref: GI:1906909115
                 โ–ผ
740422..741672  [adeI] โ—„โ—„โ—„โ—„โ—„โ—„ H0N29_03550 ๐Ÿ”ด FULLY DELETED
                 โ”‚ Product: multidrug efflux RND transporter periplasmic adaptor subunit AdeI
                 โ”‚ Length: 1,251 bp (417 aa); Protein ID: QNT85686.1
                 โ”‚ โš ๏ธ Deletion end: 741667 → ~5 bp of 3′ end preserved
                 โ–ผ
741697..742323  [PAP2 phosphatase] H0N29_03555 (downstream, intact)
                 โ”‚ Product: phosphatase PAP2 family protein; Protein ID: QNT85687.1

Functional Impact Summary:
• AdeJ (permease) + AdeI (adaptor) complete loss → tripartite RND pump cannot assemble
• adeK 3′ truncation → likely unstable/nonfunctional outer membrane channel
• Phenotypic consequence: potential increased susceptibility to AdeIJK substrates:
  - Aminoglycosides (amikacin, gentamicin, tobramycin)
  - Fluoroquinolones (ciprofloxacin, levofloxacin)
  - β-lactams (cefepime, imipenem, meropenem)
  - Tetracyclines (tigecycline, minocycline)
  - Chloramphenicol, trimethoprim
• Compensatory mechanisms: Other efflux systems (AdeABC, AdeFGH, AbeM) may be upregulated

๐Ÿ”ด SV_adeAB_del: AdeAB Efflux Pump Deletion (1844323โ€“1848605)

Reference structure (forward strand →):

1844319..1845509  [adeA] โ–บโ–บโ–บโ–บ H0N29_08675 ๐Ÿ”ด FULLY DELETED
                   โ”‚ Product: multidrug efflux RND transporter periplasmic adaptor subunit AdeA
                   โ”‚ Length: 1,191 bp (397 aa); Protein ID: QNT86625.1
                   โ”‚ โš ๏ธ Deletion start: 1844323 → 4 bp of 5′ end preserved (likely nonfunctional)
                   โ–ผ
1845506..1848616  [adeB] โ–บโ–บโ–บโ–บ H0N29_08680 ๐Ÿ”ด FULLY DELETED
                   โ”‚ Product: multidrug efflux RND transporter permease subunit AdeB
                   โ”‚ Length: 3,111 bp (1,037 aa); Protein ID: QNT86626.1
                   โ”‚ โš ๏ธ Deletion end: 1848605 → ~11 bp of 3′ end preserved (nonfunctional)
                   โ–ผ
1848764..1851025  [H0N29_08685] โ—„โ—„โ—„โ—„
                   โ”‚ Product: excinuclease ABC subunit UvrA; Protein ID: QNT86627.1
                   โ”‚ Status: โœ… INTACT (starts 159 bp downstream)

Upstream regulatory genes (INTACT):
• adeS (H0N29_08665): two-component sensor histidine kinase AdeS (1842325–1843398)
• adeR (H0N29_08670): efflux system response regulator transcription factor AdeR (1843430–1844173)

Functional Impact Summary:
• AdeA (adaptor) + AdeB (permease) complete loss → tripartite RND pump cannot assemble
• Phenotypic consequence: potential increased susceptibility to AdeAB substrate antibiotics:
  - Aminoglycosides, fluoroquinolones, β-lactams, tetracyclines, chloramphenicol
• Regulatory paradox: adeS/adeR intact but structural genes deleted → possible compensatory evolution or pseudogenization over time

๐ŸŸข SV_tRNA_contract: tRNA-Gln Array Contraction (3124916โ€“3125037)

Reference tRNA-Gln tandem array (forward strand →):

3124675..3124749  [tRNA-Gln #1] H0N29_14850 (75 bp) โ”‚ anticodon: ttg (CAG/CAA)
3124841..3124915  [tRNA-Gln #2] H0N29_14855 (75 bp) โ”‚ anticodon: ttg
3124916..3124942  [spacer] (27 bp)
3124943..3125017  [tRNA-Gln #3] H0N29_14860 (75 bp) ๐Ÿ”ด LOST in contraction
                   โ”‚ Product: tRNA-Gln; anticodon: ttg; inference: tRNAscan-SE:2.0.4
3125018..3125037  [spacer] (20 bp)
3125039..3125113  [tRNA-Gln #4] H0N29_14865 (75 bp) โ”‚ anticodon: ttg

Functional Impact Summary:
• Copy number reduction: 4 → 3 identical tRNA-Gln genes
• Likely neutral: tRNA genes are highly redundant in bacteria; single copy loss rarely affects translation efficiency
• Utility: Stable molecular marker for:
  - Clonal tracking across experiments
  - Quality control (present in 100% of high-quality assemblies)
  - Phylogenetic analysis (fixed in this lineage)

๐ŸŸข SV_flu_tandem: Intergenic Tandem Contraction (2259736โ€“2260384)

Reference context:

2216347..2217351  [cydB] โ—„โ—„โ—„โ—„ H0N29_10425 โ”‚ Cytochrome bd oxidase subunit II (ends at 2217351)
                   โ”‚ Product: cytochrome bd ubiquinol oxidase subunit II; EC: 7.1.1.7
                   โ”‚ Protein ID: QNT85688.1; DBxref: GI:1906909118
                   โ”‚ Status: โœ… INTACT (~42 kb upstream of contraction)
                   โ–ผ
2259736..2260384  [๐Ÿ”ด CONTRACTION REGION: ~135 bp]
                   โ”‚ No annotated protein-coding CDS in GFF3 excerpt
                   โ”‚ Likely repetitive element (IS, CRISPR, prophage remnant, or low-complexity DNA)
                   โ”‚ Assemblytics metrics: ref_gap: -648; query_gap: -780

Functional Impact Summary:
• No CDS disruption → likely neutral at protein level
• Possible regulatory impact if contraction removes:
  - Promoter/enhancer elements affecting downstream genes
  - Small RNA genes or riboswitches
  - DNA topology elements affecting local chromatin structure
• Utility: Condition-specific marker for fluoroquinolone selection (nitro/polyB/tet)

๐ŸŸก SV_mito_tandem: Large Repeat Array Contraction (2494563โ€“2536071)

Reference context (genes/features within/adjacent to region):

2474459..2476600  [H0N29_11610] โ—„ hypothetical protein (upstream boundary)
                   โ”‚ Product: hypothetical protein; Protein ID: QNT83948.1
                   โ”‚ Status: โš ๏ธ Partial overlap at 3′ end
                   โ–ผ
2476597..2477610  [H0N29_11615] โ—„ pseudo CDS, hypothetical protein
                   โ”‚ Note: incomplete; partial on complete genome; missing start
                   โ”‚ Status: โš ๏ธ Partial overlap
                   โ–ผ
2494563..2536071  [๐Ÿ”ด CONTRACTION REGION: 41,564 bp]
                   โ”‚ Multiple hypothetical proteins in H0N29_11xxx series (partial/full overlap)
                   โ”‚ Transposase/integrase remnants (mobile element-associated)
                   โ”‚ Repeat-rich, low-complexity sequence
                   โ”‚ Assemblytics metrics: ref_gap: 41508; query_gap: -56

Functional Impact Summary:
• Large repeat array contraction → possible loss of mobile element-associated sequences
• Hypothetical proteins affected → functional impact unknown; likely non-essential
• Hypothesis: Mitomycin C (DNA crosslinker) induces replication fork collapse → error-prone repair → large contractions in repetitive regions
• Utility: Marker for mitomycin-selected lineage; dAB-background specific

๐ŸŸก SV_mito_large_del2: Large Deletion Affecting Methyltransferase (2621714โ€“2664046)

Reference context (genes overlapping region):

2626228..2626773  [H0N29_12335] โ—„ hypothetical protein ๐Ÿ”ด FULLY DELETED
                   โ”‚ Product: hypothetical protein; Protein ID: QNT83848.1
                   โ”‚ Length: 546 bp (182 aa)
                   โ–ผ
2655635..2656549  [H0N29_12525] โ—„ DNA cytosine methyltransferase ๐Ÿ”ด FULLY DELETED
                   โ”‚ Product: DNA cytosine methyltransferase; EC: 2.1.21.-
                   โ”‚ Protein ID: QNT83886.1; DBxref: GI:1906907316
                   โ”‚ Length: 915 bp (305 aa)
                   โ”‚ Function: Catalyzes methylation of cytosine residues in DNA; epigenetic regulation

Functional Impact Summary:
• H0N29_12525 (DNA cytosine methyltransferase) loss → potential epigenetic regulation changes:
  - Altered DNA methylation patterns
  - Possible effects on gene expression, phase variation, or restriction-modification systems
• H0N29_12335 (hypothetical) loss → functional impact unknown; likely neutral
• Hypothesis: Mitomycin-induced genomic instability → adaptive genome reduction in non-essential regions
• Utility: Marker for mitomycin-selected lineage (all mito_* samples)

๐ŸŸก SV_mito_large_del1: Large Gene-Sparse Deletion (1189156โ€“1236440)

Reference context (genes/features within/adjacent to region):

1168871..1169884  [lipA] โ—„โ—„โ—„โ—„ H0N29_05320 โ”‚ Lipase (upstream, intact)
                   โ”‚ Product: lipase; EC: 3.1.1.3; Protein ID: QNT85998.1
                   โ–ผ
1171737..1172951  [H0N29_05330] โ—„ hypothetical protein (upstream, intact)
                   โ”‚ Product: hypothetical protein; Protein ID: QNT85999.1
                   โ–ผ
~1188xxx          [H0N29_05785] โ—„ tRNA-Arg (near 5′ boundary) โš ๏ธ potentially affected
                   โ”‚ Product: tRNA-Arg; inference: tRNAscan-SE
                   โ”‚ Status: Boundary proximity; possible regulatory element loss
                   โ–ผ
1189156..1236440  [๐Ÿ”ด DELETION REGION: 47,299 bp]
                   โ”‚ Gene-sparse region
                   โ”‚ Multiple hypothetical proteins in H0N29_05xxx series (partial/full overlap)
                   โ”‚ Possible non-essential genomic island or prophage remnant
                   โ–ผ
1235097..1235492  [H0N29_05775] โ—„ hypothetical protein (partial overlap at 3′ boundary)
                   โ”‚ Product: hypothetical protein; Protein ID: QNT86000.1
                   โ–ผ
1263952..1264122  [H0N29_05915] โ—„ hypothetical protein (downstream, intact)
                   โ”‚ Product: hypothetical protein; Protein ID: QNT86001.1

Functional Impact Summary:
• tRNA-Arg near boundary: deletion may affect tRNA expression/regulation if promoter elements removed
• Multiple hypothetical proteins lost → likely non-essential functions
• Hypothesis: Adaptive genome reduction under mitomycin stress; loss of non-essential functions to reduce metabolic burden
• Utility: Marker for mitomycin + dAB background lineage

๐Ÿ“‹ Export-Ready Comprehensive Annotation Table (TSV Format)

SV_ID   Coordinates Type    Size_bp Affected_Genes_LocusTags    Affected_Genes_Products Overlap_Type_per_Gene   Functional_Impact_Summary   Sample_Pattern  Priority
SV_adeIJ_del    737224-741667   Deletion    4436    adeK(H0N29_03540);adeJ(H0N29_03545);adeI(H0N29_03550)   multidrug_efflux_RND_transporter_subunits_AdeIJK    adeK:3prime_truncation;adeJ:full_deletion;adeI:full_deletion    Loss_of_AdeIJK_pump_function;potential_increased_antibiotic_susceptibility  all_dIJ_samples HIGH
SV_adeAB_del    1844323-1848605 Deletion    4282    adeA(H0N29_08675);adeB(H0N29_08680) multidrug_efflux_RND_transporter_subunits_AdeAB adeA:full_deletion_4bp_5prime_preserved;adeB:full_deletion_11bp_3prime_preserved    Loss_of_AdeAB_pump_function;potential_increased_antibiotic_susceptibility   all_dAB_samples HIGH
SV_tRNA_contract    3124916-3125037 Tandem_contraction  198 tRNA-Gln(H0N29_14860)   tRNA-Gln_anticodon:ttg_glutamine_codon_translation  H0N29_14860:full_deletion_75bp  tRNA_dosage_reduction_4to3;likely_neutral_lineage_marker    all_filtered_samples    LOW
SV_flu_tandem   2259736-2260384 Tandem_contraction  135 intergenic_no_CDS_overlapped    Likely_neutral_repetitive_element   No_CDS_disruption;possible_regulatory_impact    Likely_neutral;fluoroquinolone_selection_marker flu_subset_condition_specific   LOW
SV_mito_tandem  2494563-2536071 Tandem_contraction  41564   H0N29_11610(partial);H0N29_11615(partial);multiple_H0N29_11xxx_hypotheticals    hypothetical_proteins;repeat_rich_region    Multiple_hypotheticals:partial_or_full_deletion;repeat_arrays:contraction   Large_repeat_array_contraction;likely_non-essential;mobile_element_associated_plasticity    mito_dAB_only   MEDIUM
SV_mito_large_del2  2621714-2664046 Deletion    42352   H0N29_12335(full);H0N29_12525(full) hypothetical_protein;DNA_cytosine_methyltransferase_EC:2.1.21.- H0N29_12335:full_deletion;H0N29_12525:full_deletion Loss_of_DNA_cytosine_methyltransferase;potential_epigenetic_regulation_changes  all_mito_samples    MEDIUM
SV_mito_large_del1  1189156-1236440 Deletion    47299   tRNA-Arg(H0N29_05785,boundary);H0N29_05775(partial);multiple_H0N29_05xxx_hypotheticals  tRNA-Arg;hypothetical_proteins  tRNA-Arg:boundary_proximity;H0N29_05775:partial_deletion;others:full/partial    Possible_loss_of_non-essential_functions;tRNA_boundary_effect_uncertain;adaptive_genome_reduction   mito_dAB_only   MEDIUM

๐Ÿ”ฌ Reproducible Validation Workflow (Command-Line)

# 1. Create BED file of SV coordinates (0-based, half-open for bedtools)
cat > sv_coords.bed << EOF
CP059040    737223  741667  SV_adeIJ_del    4436    Deletion
CP059040    1844322 1848605 SV_adeAB_del    4282    Deletion
CP059040    3124915 3125037 SV_tRNA_contract    198 Tandem_contraction
CP059040    2259735 2260384 SV_flu_tandem   135 Tandem_contraction
CP059040    2494562 2536071 SV_mito_tandem  41564   Tandem_contraction
CP059040    2621713 2664046 SV_mito_large_del2  42352   Deletion
CP059040    1189155 1236440 SV_mito_large_del1  47299   Deletion
EOF

# 2. Intersect with GFF3 annotation (requires bedtools)
bedtools intersect -a sv_coords.bed -b CP059040.gff3.txt -wa -wb -loj > sv_gene_overlap.tsv

# 3. Extract and summarize affected genes per SV
awk -F'\t' 'NR>1 {print $4, $10, $11, $12}' sv_gene_overlap.tsv | \
  sort | uniq -c | column -t > sv_gene_summary.txt

# 4. Extract sequences for breakpoint validation
while IFS=$'\t' read -r chr start end sv_id size sv_type; do
  samtools faidx bacto/CP059040.fasta ${chr}:${start}-${end} > ${sv_id}_region.fasta
done < sv_coords.bed

๐Ÿ’ก Manuscript Interpretation Guidelines

High-Priority Findings (Results Section)

“Two mutually exclusive ~4.3-kb deletions define efflux pump backgrounds: SV_adeIJ_del (CP059040:737224โ€“741667) abolishes the AdeIJK pump (adeJ, adeI, truncated adeK) in all dIJ strains, while SV_adeAB_del (CP059040:1844323โ€“1848605) abolishes the AdeAB pump (adeA, adeB) in all dAB strains. Both variants result in complete loss of permease and adaptor subunits, likely conferring increased susceptibility to respective substrate antibiotics.”

Medium-Priority (Supplementary/Discussion)

“Mitomycin C-selected strains harbor large (>40 kb) structural variants in repeat-rich genomic regions (SV_mito_tandem, SV_mito_large_del1/2), absent in fluoroquinolone-selected isolates. Notably, SV_mito_large_del2 deletes a DNA cytosine methyltransferase (H0N29_12525), suggesting potential epigenetic adaptation under genotoxic stress.”

Low-Priority (Methods/QC)

“A universal 198-bp tandem contraction in a tRNA-Gln array (SV_tRNA_contract; copy number 4โ†’3) and a condition-specific ~135-bp intergenic contraction (SV_flu_tandem) were detected, serving as stable lineage markers and selection-condition signatures, respectively.”

  • BED/GFF3 files for IGV visualization of all 7 SVs with gene tracks
  • Integration with SNP/InDel results for a unified variant report
  • R/Python scripts to generate publication-ready figures (SV distribution, genome maps, gene impact heatmaps)

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