Selection, drift and bias shape bacterial peptide evolution

A 2026 evolutionary study of an insect-killing bacterial peptide family found that its chemical diversity was shaped by natural selection, genetic drift and biased recombination. The researchers reconstructed the history of GameXPeptides, or GXPs, produced by GxpS synthetases in the bacterial genera Photorhabdus and Xenorhabdus. Their analysis identified dozens of previously undescribed peptides, including structures that evolved independently more than once, while experiments and statistical comparisons showed that the historical pattern was consistent with both adaptive and non-adaptive processes.

The investigation focused on GXPs, which entomopathogenic bacteria use to kill insect prey. The GxpS genes contain repeated domains whose separate histories are difficult to reconstruct because whole genes can undergo duplication or horizontal transfer, while individual domains and modules can also be duplicated, deleted, inserted or recombined. Conventional gene trees can therefore conflate several events. To establish a reference history, the researchers used the terminal thioesterase domain together with the adjacent T domain. Recombination-aware analyses detected no recombination in these regions among the sampled GxpS sequences, allowing them to serve as an anchor for the whole gene and for the individual A- and TC-domain trees.

Analysis of 54 putative GxpS orthologues placed them in a monophyletic group and included the four-modular Xenotetrapeptide synthetase and an unknown six-modular non-ribosomal peptide synthetase within that group. Reconciliation of the TTE-domain gene tree with a species tree indicated that GxpS first arose in Photorhabdus and was subsequently transferred to Xenorhabdus twice. This conflict between gene and species histories was interpreted as evidence of horizontal gene transfer. The researchers also traced the separate operon that supplies p-aminophenylalanine and its methylated derivative, concluding that it was present in the last common ancestor of the analyzed bacteria, was lost twice in Xenorhabdus groups, and was reacquired by horizontal transfer in Xenorhabdus doucetiae. In that species, the operon contains mobile genetic elements and experiments indicated that it was probably inactive.

Disagreement among the trees of the other GxpS domains exposed extensive recombination. Some exchanges replaced entire adenylation domains, whereas others transferred short sections, including fragments inserted into regions already altered by an earlier event. The team predicted 17 A-domain recombinations and used a mixture of phylogenetic incongruence, local sequence similarity and recombination-detection analysis to resolve more complicated histories. Their workflow separated unrecombined sequence from donor-derived segments and identified the likely donor clade for each inferred event. Experimental validation then included expression in Escherichia coli, high-performance liquid chromatography coupled to mass spectrometry, feeding with isotopically labelled amino acids, and co-injection of chemically synthesized peptides.

Those experiments confirmed every recombination that had been predicted to create a new peptide. They also established functional consequences that could not have been read directly from sequence alone. In one example, an A2-domain fragment replaced the A3 domain of the synthetase from Xenorhabdus sp. PB62.4, changing incorporation of phenylalanine to leucine alone. Elsewhere, a fifth A-domain fragment was inserted into the middle of a third A-domain and shifted production toward a different major peptide. A five-modular enzyme produced rare cyclic tetrapeptides despite retaining five modules. Disabling its terminal T and thioesterase domains did not eliminate the product, while replacing the recombined C4 domain with an LCL-C2 domain changed the output to cyclic pentapeptides. These results showed that the recombined C4 domain enabled early release and cyclization of tetrapeptides.

Across the family, the researchers predicted at least 12 new main products and detected 49 compounds, including side products, during the experimental analyses. They identified ten unique GxpS variants with product spectra expected to differ from the canonical ortholog. Isotopic feeding and peptide co-injection supported the proposed structures and substrate incorporation. They also found that every tested phenylalanine-incorporating synthetase could incorporate p-aminophenylalanine or its methylated derivative; in almost all feeding tests, the resulting peptide was dominant under the conditions used. For Xenorhabdus species lacking the operon, the phenylalanine-containing GameXPeptide was the main product. The scale of the recovered family diversity therefore extended beyond the canonical peptide, even though the study examined synthetases from only two bacterial genera.

Ancestral sequence reconstruction offered another view of the diversification. The researchers reconstructed the last common ancestor of the analyzed GxpS family, assembled its domains into a complete synthetase and expressed it in Escherichia coli. The inferred ancestor produced a cyclic pentapeptide with a structure similar to that of the canonical modern peptide. A deliberately less probable reconstruction, differing at 244 amino-acid positions, produced the same major product. Mapping peptide structures onto the TTE-domain tree revealed extensive convergence: phenylalanine at the third position was independently replaced by leucine seven times, producing four synthetases with the same major peptide, while 12 independent recombinations introduced valine-specific domains and yielded five origins of another pentapeptide. Tetrapeptide production arose through several routes, including two independently evolved four-modular synthetases and five-modular enzymes that acquired an early-cyclization domain.

Laboratory feeding tests then showed that the structural differences among GXPs translated into different insecticidal activities. The researchers tested eight peptides against larvae of Spodoptera exigua, Plutella xylostella, Tenebrio molitor and Frankliniella occidentalis. Each treatment contained 10 larvae, each was conducted with three biological replicates, and mortality was recorded three days later. Comparison of five models by the Akaike information criterion favored the most complex model, which assigned a distinct potency to each peptide–insect combination. Peptide 36 was the most active against S. exigua but among the least effective against P. xylostella, whereas peptide 4 was most effective against P. xylostella. The experiment nevertheless covered only four insect species, and the authors cautioned against assuming that any one GXP exclusively targets a particular insect in nature.

Recombination statistics supplied evidence that selection affected some events. The researchers distinguished homotypic exchanges, which involved domains with the same substrate specificity and did not change the peptide’s composition, from heterotypic exchanges involving different specificities. Homotypic events served as a proxy for the neutral process. Heterotypic recombinations affected the specificity pocket of the A-domain significantly more often than expected from that proxy; Fisher’s exact tests gave P less than 0.05 in the evaluated tables. This pattern was consistent with rare, specificity-changing events being preferentially retained by selection. It was not a direct measurement of the fitness of ancient peptides, however, and the authors also described a possible non-selective explanation. The 27 synonymous recombination events identified, compared with 26 non-synonymous events, suggested that drift also played a substantial role in GxpS diversification. Likewise, the presence of several peptides with similar potency against each tested insect left room for chemically distinct variants to persist without a large measured activity difference.

A third influence emerged from donor bias. The actual historical A-domain donors were generally more similar at the nucleotide level to their recipients than alternative donors with the same specificity located elsewhere in the same genome. Candidate donors from other non-ribosomal peptide synthetase genes were significantly less similar, with P less than 10⁻⁴ in the relevant paired comparisons. Most A-domain donors also came from within GxpS itself. This pattern suggests a self-reinforcing restriction: recombination among similar domains within the gene would homogenize portions of the sequence and make later exchanges among those internal domains still more likely, while less-similar external domains would remain less accessible. The observed convergence may therefore reflect both the benefits of a particular peptide and the limited set of substitutions that biased recombination made readily available.

Together, the results portray GxpS evolution as the outcome of interacting forces rather than a purely adaptive or purely random process. Selection was consistent with preferential retention of some specificity-changing recombinations, drift accompanied many neutral and nearly neutral structural changes, and recombination bias restricted which amino-acid specificities could be accessed easily. The study also has clear limits. It examined one synthetase family, tested only eight peptides and four insect species, and used homotypic recombination as an approximation to neutrality. Synonymous events were harder to verify experimentally, while some could still have affected peptide production or enzyme folding. The authors further noted that every recombination overwrites part of the sequence record, meaning that some ancient history may be unrecoverable. Whether the same combination of forces shapes other non-ribosomal peptide families remains an open question that will require analyses of other synthetase systems.

Subject of Research: Evolutionary drivers of non-ribosomal peptide diversification in entomopathogenic bacteria

Article Title: Drift, selection and recombination bias drive the evolution of a non-ribosomal peptide

Article References: Kaiser, S., Shahmohammadi, N., Goel, T. S. M., Buchanan, C. J., Garg, S. G., Vidaurre-Barahona, D., Qian, Z., Lefèvre, C., Schindler, D., Kim, Y., Bode, H. B., & Hochberg, G. K. A. (2026). Drift, selection and recombination bias drive the evolution of a non-ribosomal peptide. Nature Ecology & Evolution. https://doi.org/10.1038/s41559-026-03214-y

Image Credits: AI Generated

DOI: 10.1038/s41559-026-03214-y

Keywords: non-ribosomal peptides, GameXPeptides, GxpS, bacterial evolution, genetic recombination, natural selection, genetic drift, convergent evolution, entomopathogenic bacteria, insecticidal peptides, Drift, selection