Horizontal gene transfer (HGT) is an important factor in bacterial evolution that can act across species boundaries. Yet, we know little about rate and genomic targets of cross-lineage gene transfer and about its effects on the recipient organism's physiology and fitness. Here, we address these questions in a parallel evolution experiment with two Bacillus subtilis lineages of 7% sequence divergence. We observe rapid evolution of hybrid organisms: gene transfer swaps ∼12% of the core genome in just 200 generations, and 60% of core genes are replaced in at least one population. By genomics, transcriptomics, fitness assays, and statistical modeling, we show that transfer generates adaptive evolution and functional alterations in hybrids. Specifically, our experiments reveal a strong, repeatable fitness increase of evolved populations in the stationary growth phase. By genomic analysis of the transfer statistics across replicate populations, we infer that selection on HGT has a broad genetic basis: 40% of the observed transfers are adaptive. At the level of functional gene networks, we find signatures of negative, positive, and epistatic selection, consistent with hybrid incompatibilities and adaptive evolution of network functions. Our results suggest that gene transfer navigates a complex cross-lineage fitness landscape, bridging epistatic barriers along multiple high-fitness paths.

Gene tandem amplifications are thought to drive bacterial evolution, but they are transient in the absence of selection, making their investigation challenging. Here, we analyze genomic sequences of Staphylococcus aureus USA300 isolates from the same geographical area to identify variations in gene copy number, which we confirm by long-read sequencing. We find several hotspots of variation, including the csa1 cluster encoding lipoproteins known to be immunogenic. We also show that the csa1 locus expands and contracts during bacterial growth in vitro and during systemic infection of mice, and recombination creates rapid heterogeneity in initially clonal cultures. Furthermore, csa1 copy number variants differ in their immunostimulatory capacity, revealing a mechanism by which gene copy number variation can modulate the host immune response.

In the stationary phase, Bacillus subtilis differentiates stochastically and transiently into the state of competence for transformation (K-state). The latter is associated with growth arrest, and it is unclear how the ability to develop competence is stably maintained, despite its cost. To quantify the effect differentiation has on the competitive fitness of B. subtilis, we characterized the competition dynamics between strains with different probabilities of entering the K-state. The relative fitness decreased with increasing differentiation probability both during the stationary phase and during outgrowth. When exposed to antibiotics inhibiting cell wall synthesis, transcription, and translation, cells that differentiated into the K-state showed a selective advantage compared to differentiation-deficient bacteria; this benefit did not require transformation. Although beneficial, the K-state was not induced by sub-MIC concentrations of antibiotics. Increasing the differentiation probability beyond the wt level did not significantly affect the competition dynamics with transient antibiotic exposure. We conclude that the competition dynamics are very sensitive to the fraction of competent cells under benign conditions but less sensitive during antibiotic exposure, supporting the picture of stochastic differentiation as a fitness trade-off.

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9Horizontal gene transfer is an important factor in bacterial evolution that can act across 10 species boundaries. Yet, we know little about rate and genomic targets of cross-lineage 11 gene transfer, and about its effects on the recipient organism's physiology and fitness. 12Here, we address these questions in a parallel evolution experiment with two Bacillus 13 subtilis lineages of 7% sequence divergence. We observe rapid evolution of hybrid 14 organisms: gene transfer swaps ~12% of the core genome in just 200 generations, and 15 60% of core genes are replaced in at least one population. By genomics, transcriptomics, 16fitness assays, and statistical modeling, we show that transfer generates adaptive evolution 17 and functional alterations in hybrids. Specifically, our experiments reveal a strong, 18repeatable fitness increase of evolved populations in the stationary growth phase. By 19 genomic analysis of the transfer statistics across replicate populations, we infer that 20 selection on HGT has a broad genetic basis: 40% of the observed transfers are adaptive. 21At the level of functional gene networks, we find signatures of negative and positive 22 selection, consistent with hybrid incompatibilities and adaptive evolution of network 23 functions. Our results suggest that gene transfer navigates a complex cross-lineage fitness 24 landscape, bridging epistatic barriers along multiple high-fitness paths. 25 26 Significance statement 27In a parallel evolution experiment, we probe lateral gene transfer between two Bacillus subtilis 28 lineages close to the species boundary. We show that laboratory evolution by horizontal gene 29 transfer can rapidly generate hybrid organisms with broad genomic and functional alterations. 30By combining genomics, transcriptomics, fitness assays and statistical modeling, we map the 31 selective effects underlying gene transfer. We show that transfer takes place under genome-32 wide positive and negative selection, generating a net fitness increase in hybrids. The 33 evolutionary dynamics efficiently navigates this fitness landscape, finding viable paths with 34 increasing fraction of transferred genes. 35 36