Analysis of bacterial genomes from an evolution experiment with horizontal gene transfer shows that recombination can sometimes overwhelm selection

Abstract

Few experimental studies have examined the role that sexual recombination plays in bacterial evolution, including the effects of horizontal gene transfer on genome structure. To address this limitation, we analyzed genomes from an experiment in which Escherichia coli K-12 Hfr (high frequency recombination) donors were periodically introduced into 12 evolving populations of E. coli B and allowed to conjugate repeatedly over the course of 1000 generations. Previous analyses of the evolved strains from this experiment showed that recombination did not accelerate adaptation, despite increasing genetic variation relative to asexual controls. However, the resolution in that previous work was limited to only a few genetic markers. We sought to clarify and understand these puzzling results by sequencing complete genomes from each population. The effects of recombination were highly variable: one lineage was mostly derived from the donors, while another acquired almost no donor DNA. In most lineages, some regions showed repeated introgression and others almost none. Regions with high introgression tended to be near the donors' origin of transfer sites. To determine whether introgressed alleles imposed a genetic load, we extended the experiment for 200 generations without recombination and sequenced whole-population samples. Beneficial alleles in the recipient populations were occasionally driven extinct by maladaptive donor-derived alleles. On balance, our analyses indicate that the plasmid-mediated recombination was sufficiently frequent to drive donor alleles to fixation without providing much, if any, selective advantage.

Fig 1. Genome structure of odd-numbered clones from recombinant populations after 1000 generations of the STLE.

The REL606 genomic coordinates are shown on the x-axis, centered on the oriC origin of replication, and the source populations are shown on the y-axis. Genetic markers are shown as vertical lines, with the color indicating the origin of each marker. Markers specific to K-12 donors are yellow; markers specific to recipient clones are blue; markers in deleted regions are light purple; new mutations that arose during the STLE are black; and LTEE-derived mutations that were replaced by donor DNA during the STLE are red. In addition, symbols indicate mutations in genes under positive selection in the LTEE (Table 1). Open circles indicate nonsynonymous point mutations; open squares are synonymous mutations; open triangles are indels; and x-marks are IS-element insertions. Replaced and new mutations in the genes in Table 1 are labeled by their gene names.

Fig 2. Impact of donors’ transfer origins and auxotrophic mutations on introgression.

(A) The number of parallel introgressions of K-12 genetic markers summed over the odd-numbered STLE clones (omitting the Ara–3 clone, which is almost completely derived from K-12 donor DNA). A natural cubic spline with 100 degrees of freedom, in blue, was fit to these data. The locations of auxotrophic mutations in the donor genomes are shown as dashed vertical lines, and the location and orientation of the oriT transfer origin sites are labeled below the x-axis. The markers and transfer origins for REL288 are shown in red, for REL291 in green, for REL296 in teal, and for REL298 in purple. The colored arrows indicate the genome regions most likely to be transferred by each Hfr. (B) Auxotrophic mutations do not cause a complete barrier to introgression. The location of donor-specific markers found in the odd-numbered recombinant clones (including Ara–3) are labeled with the same colors as in panel (A). Note that almost all of the introgressions near an auxotrophic mutation in one donor strain came from a different donor that did not carry that mutation.

Fig 3. Protein alignments containing new alleles generated via recombination or by subsequent mutation in odd-numbered STLE recombinant clones.

Only variable sites are shown. Red columns show K-12 markers introduced by a recombination event. (A) Ara–1 yghJ: this locus appears to have experienced at least 3 recombination events. (B) Ara–4 pykF: the recombinant is missing a T462I mutation present in the recipient, and it has a new D127N mutation. (C) Ara+1 hslU: the recombinant is missing an E47G substitution found in the recipient, and it has a new L395V mutation. (D). Ara+3 nfrA: the recombinant lacks the W289* nonsense mutation present in the recipient. This putative recombination event did not affect the recipient’s C144R mutation, nor did it introduce any of the K-12 markers found at residue 364 and beyond. The Ara+3 lineage is hypermutable, and the reversion of the nonsense mutation might have occurred without a recombination event.

Fig 4. The length distributions, shown on a logarithmic scale, of DNA segments derived from donors (left column) and recipients (right column) in the odd-numbered recombinant genomes.

Fig 5. The number and provenance of donor-specific genetic markers in recombinant clones.

REL288 markers are shown in red, REL291 markers in green, REL296 markers in teal, and REL298 markers in purple. Four clones have very little or no donor DNA, and another has no donor-specific markers.

Fig 6. Alleles derived from the K-12 donors tended to increase in frequency during the STLE continuation experiment.

Initial and final K-12 allele frequencies are plotted on the x- and y-axis, respectively. Alleles that increased in frequency lie above the dashed diagonal line, and those that decreased lie below the diagonal. Alleles are colored based on their genomic position, so clusters with the same color probably belong to the same haplotype.

Fig 7. Mutations at 100% frequency in both the initial (1000 generation) and final (1200 generation) samples of the STLE continuation experiment and in both recombinant clones, which we infer to have been present in the last common ancestor (LCA) of each population.

See legend to Fig 1 for description of symbols and labels.