Status
Preliminary — core analyses complete (NB01–NB04). NB05 threshold validation and NB06 manuscript figures pending.
Key Findings
- 21.8% of 30,497 high-quality environmental MAGs carry T4SS/conjugative machinery (6,652 MAGs; multi-marker definition: VirB4/6/8/9/10/11, VirD4, TraI/D, TrwB, TraG).
- 92 CAZy families show elevated co-occurrence with T4SS loci at ≤10 kb (threshold validation pending); GT2 glycosyltransferases are the top hit (767 genomes, avg 5,041 bp).
- Biome enrichment: marine sediment (OR=5.5, q<10⁻⁹⁸), barley rhizosphere (OR=10.4), maize rhizosphere (OR=4.1).
- 77 HGT events detected in GT2 gene tree; 32 high-confidence cross-phylum events (normalised). Strongest: Node_4915 spans 8 phyla at max divergence 4.843.
- CAZy genes are not on plasmids (ICEfinder: 12 IMEs in top 100 accumulators); T4SS+ genomes have 10× higher MGE density (p<0.001), consistent with chromosomal/integrative transfer.
- Discovery/validation split (70/30): enrichment patterns replicate across sets, confirming robustness.
Interpretation
T4SS machinery in environmental MAGs is significantly co-localised with GT2 glycosyltransferase cassettes, and phylogenetic incongruence in the GT2 gene tree provides positive evidence for 32 cross-phylum HGT events. The ICEfinder null result (no plasmid-borne CAZy genes) combined with the 10× MGE density elevation in T4SS+ genomes is consistent with chromosomal or integrative transfer mechanisms (IMEs/ICEs) rather than plasmid mobilisation.
These results support the hypothesis that T4SS machinery mediates environmental dissemination of carbohydrate-active enzyme diversity across phylogenetically distant bacteria. All associations are observational; mechanistic confirmation requires experimental validation.
NB05 Analysis Results
HGT event characterisation (Detected_HGT_Events.csv, n=77):
- Distinct_Phyla distribution: 65 events span 2 phyla, 12 span ≥3 phyla (15.6%).
- Node_4915 confirmed: 35 genes, 82.9% syntenic, 8 phyla (WOR-3, Desulfobacterota, Patescibacteria, Bacteroidota, Firmicutes_A, Methanobacteriota, Bdellovibrionota, Acidobacteriota), Max_Divergence = 4.843.
- Divergence vs. synteny: Spearman ρ = −0.615 (p<0.001) — more phylogenetically distant events have lower syntenic percentage, consistent with sequence divergence after transfer. This is the expected biological pattern.
- Most-involved phyla across all events: Firmicutes_A (27), Pseudomonadota (22), Bacillota_A (19), Actinomycetota (10).
- Figure:
figures/fig_nb05_hgt_scatter.png
GT2 neighbourhood analysis (376 genomes):
- Neighbourhood parsed as list-format: T4SS co-occurs in 503 neighbourhood entries, GT2 in 495 — confirming syntenic co-localisation at the contig level.
- GH23 (murein lytic transglycosylase) is the second most common CAZy family in GT2 neighbourhoods (106 occurrences), suggesting cell wall remodelling genes cluster with GT2-T4SS syntenic loci.
- Figure:
figures/fig_nb05_neighbourhood_functions.png
GT2 × metal resistance (new finding): GT2-neighbourhood MAGs (n=376) have mean 0.045 metal resistance types vs 0.004 for non-GT2 MAGs (n=260,276); Mann-Whitney p=8.6e-27. Genomes with GT2 in T4SS-proximal neighbourhoods carry 11× more metal resistance genes. This is an independent link between CAZy-T4SS synteny and the metal resistance niche breadth hypothesis: genomes that serve as hubs for GT2 horizontal transfer are also enriched for metal resistance functions.
Pending Validation
- Synteny threshold permutation test (requires Spark unfiltered data)
- Node_4915 BLAST validation (requires BLAST against NCBI nr)
- Housekeeping gene null baseline
- Biome enrichment factorisation: θ = OR(T4SS-CAZy) / [OR(T4SS) × OR(CAZy)]