Horizontal gene transfer events between bacteria and eukaryotes detected through comparative genomics

How do bacterial genes end up inside eukaryotic chromosomes, and how often does this actually happen? This research applied a comparative genomics pipeline to 318 eukaryotic genome assemblies to detect and validate horizontal gene transfer (HGT) events originating from bacterial donors. The analysis, conducted at the Instituto de Biología Marina de Canarias between February 2022 and November 2023, combined sequence similarity screening with phylogenetic incongruence testing, GC content analysis, and intron pattern verification. A total of 187 high-confidence HGT events were identified across six eukaryotic phyla. Proteobacteria were the most frequent donors (36 events), and fungi were the most common recipients (47 events). Transferred genes were enriched for metabolic functions, particularly amino acid biosynthesis and xenobiotic degradation. These results indicate that bacteria-to-eukaryote HGT is more widespread than previously estimated and has shaped metabolic capacity across diverse eukaryotic lineages.

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HGT Detection Pipeline FlowClose ×

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Fungi and Arthropoda and more: Proteobacteria vs Firmicutes vs Actinobacteria vs Bacteroidetes vs CyanobacteriaClose ×

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Introduction

The assumption that genes travel strictly from parent to offspring doesn't hold for all branches of the tree of life. Horizontal gene transfer-the movement of genetic material between organisms outside of vertical inheritance-is well documented among bacteria, where it drives antibiotic resistance and metabolic diversification [1] . But the extent of HGT from bacteria to eukaryotes has been debated for decades, with estimates ranging from negligible to surprisingly common depending on the methods used [2] . Part of the difficulty is methodological. Contamination of genome assemblies with bacterial sequences can mimic HGT, and incomplete lineage sorting can produce phylogenetic patterns that resemble transfer events [3] . Reliable detection requires multiple lines of evidence: unusual phylogenetic placement, atypical sequence composition (such as GC content), and structural features like the absence of introns in a gene that belongs to an intron-rich family [4] . This research developed a multi-filter pipeline and applied it to 318 publicly available eukaryotic genomes at the Instituto de Biología Marina de Canarias, Las Palmas, Spain, between February 2022 and November 2023 [5] .

Theoretical Background

The theoretical basis for detecting HGT rests on the expectation that vertically inherited genes produce phylogenetic trees congruent with the species tree, while horizontally transferred genes produce incongruent topologies [6] . The approximately unbiased (AU) test provides a statistical framework for rejecting the null hypothesis of vertical inheritance when a gene tree differs significantly from the species tree [7] . Complementary evidence comes from compositional analysis: recently transferred bacterial genes often retain a GC content closer to their donor genome than to the recipient eukaryote, though amelioration erases this signal over evolutionary time [4] . Intron patterns offer a third diagnostic: bacterial genes inserted into eukaryotic genomes initially lack introns, and intron gain occurs gradually [8] .

Material and Methods

Material and Methods

Material

Genome assemblies for 318 eukaryotic species (87 fungi, 63 arthropods, 48 nematodes, 42 chordates, 51 plants, 27 amoebozoans) were retrieved from NCBI GenBank (accessed March 2022). Bacterial reference proteomes (5,124 species) were obtained from UniProt Reference Proteomes release 2022_01. All computational work was performed on the high-performance computing cluster at the Instituto de Biología Marina de Canarias, Las Palmas, Canary Islands, Spain.

Methods

Predicted protein sequences from each eukaryotic genome were screened against the bacterial reference proteomes using DIAMOND BLASTp (E-value < 1e-10, query coverage >70%) [9] . Candidate HGT genes were required to have their best BLAST hit in a bacterial taxon and no close homologs in related eukaryotic lineages. Phylogenetic trees were built with IQ-TREE v2.2 using the best-fit substitution model selected by ModelFinder [10] . The AU test was applied to compare gene tree topology against the expected species tree. Candidates passing phylogenetic screening were further filtered by GC content deviation (>5% from the recipient genome mean) and intron count analysis (zero or fewer introns than the family average). Functional annotation used InterProScan v5.59 and KEGG pathway mapping. Statistical enrichment was assessed by Fisher's exact test with Benjamini-Hochberg correction.

Performance Evaluation: The pipeline was benchmarked against a curated set of 42 previously published bacteria-toeukaryote HGT events [2] . Sensitivity was 88.1% (37 of 42 recovered), and specificity was assessed by running 200 simulated vertical inheritance genes through the pipeline, yielding a false-positive rate of 3.5%. Missed events were cases where donor lineages had diverged beyond detectable sequence similarity. Processing time averaged 4.2 hours per genome on a 64-core node with 256 GB RAM. A total of 187 HGT events passed all four filters. Fungi were the most frequent recipients (47 events), followed by nematodes (32) and arthropods (28). Cyanobacterial transfers to plants dominated one donor-recipient pair (14 events), reflecting the known history of plastid-associated gene movement [11] .

Results

Results

Discussion

The 187 confirmed HGT events identified here suggest that bacteria-to-eukaryote gene transfer is more common than early genome-era estimates indicated. The enrichment of transferred genes in metabolic pathways-particularly amino acid biosynthesis and xenobiotic degradation implies that HGT confers adaptive advantages by expanding the biochemical repertoire of recipient organisms [12] . Fungi emerged as the most receptive lineage, possibly because their close ecological contact with soil bacteria creates frequent opportunities for DNA uptake [13] .

The high rate of cyanobacterial transfers to plants (14 events) likely reflects ongoing gene movement from plastidassociated bacterial genomes, a well-documented phenomenon [11] . A key limitation is that our pipeline may undercount ancient transfer events where amelioration has erased compositional signatures and sequence divergence has weakened phylogenetic signals [14] . Future work could incorporate synteny conservation and chromatin context to improve detection of older HGT events. The multi-filter approach used here provides a reproducible framework that can be applied as new genome assemblies become available [15] .

Conclusion

This research identified 187 high-confidence horizontal gene transfer events from bacteria to eukaryotes across 318 genome assemblies. A four-filter detection pipeline achieved 88.1% sensitivity and a 3.5% false-positive rate against curated benchmarks. Transferred genes were enriched for metabolic functions, and fungi were the most frequent recipients. These findings support the view that interkingdom gene transfer has been a recurring force in eukaryotic genome evolution and metabolic adaptation.

Acknowledgement

Acknowledgements Funding SourcesThis research was supported by the Instituto de Biología Marina de Canarias internal research programme. Institutional SupportThe authors thank the Department of Systematics and Evolution for access to high-performance computing resources. Contributions Not Qualifying for AuthorshipSystem administrators at the Instituto maintained the computational cluster used for all analyses.

Figures and tables from the paper

Donor Phylum	Fungi	Arthropoda	Nematoda	Chordata	Plantae	Amoebozoa
Proteobacteria	12	8	6	3	9	5
Firmicutes	7	4	11	2	3	4
Actinobacteria	5	3	4	1	7	2
Bacteroidetes	9	6	3	1	2	8
Cyanobacteria	3	2	1	0	14	1

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Distribution of Confirmed HGT Events by Donor and Recipient Lineage

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Heatmap showing the frequency of confirmed HGT events between bacterial donor phyla and eukaryotic recipient lineages.

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Multi-filter pipeline for HGT detection, from initial BLAST screening through phylogenetic incongruence testing, compositional

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Original paper figureSource PDF · page 3 ↗

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