Drift, selection, and convergence in the evolution of a nonribosomal peptide
Abstract
Nonribosomal peptide synthetases (NRPS) produce peptides with large structural
diversity. These secondary metabolites play an important role in microbial ecology, for
example these metabolites can have antibiotic properties to kill bacterial competitors.
For humans, nonribosomal peptides (NRP) play a crucial role because many of these
products are used as important clinical drugs. Examples of such products are the
antibiotics penicillin and vancomycin. NRPS usually consist of repeating modules that
are CAT-tridomains. The Condensation (C)-domain facilitates peptide bond formation,
the Adenylation (A)-domain selects and activates the incorporated amino acid and the
Thiolation (T)-domain carries the incorporated amino acids. How and why evolution
has shaped NRPS is not understood in detail. However, it is known that NRPS mainly
change their corresponding peptide through recombination instead by mutation.
Recombination is a process where one DNA strand replaces another DNA strand.
Recombination has a dramatic effect on the genetic history of the NRPS. Each
recombination event introduces a new phylogenetic layer that leads to NRPS with
complex phylogenetic histories. This work aims to overcome this phylogenetic problem
by disentangling the complex phylogenetic history of GameXPeptide synthetase
(GxpS) using recombination-aware methods. GxpS is the most conserved NRPS
within the entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus
(XP). Its product GameXPeptide (GXP) functions as an insecticide; therefore, it plays
a crucial role in the life cycle of XP and thus GxpS is a suitable model to study NRPS
evolution.
In chapter one, I established NRPS orthology by using the Thiolation/ Thioesterase
(TTE)-domain as a gene tree. Hence, I was able to classify which NRPS belongs to
the GxpS family and at the same time, I was able to polarize the GxpS history.
Simultaneously, I was able to conduct reconciliation experiments to analyze whole
gxpS gene transfer.
In chapter two, I investigated the diversity within the family of GxpS. I did this by
inferring individual A- and TC-domain trees and applying recombination-aware
methods to detect A- and TC-domain recombination that has led to new GxpS variants.
If a clade within an A- and TC-domain deviated from the reference tree (the TTE
domain), it was always a sign of a further recombination event that facilitated untangling the complex phylogenetic history of GxpS. Through my recombination
aware analysis, I was able to predict new GXP, and I confirmed my predictions via
heterologous expression in Escherichia coli.
In chapter three, I followed in detail the trajectory of GxpS evolution using ancestral
sequence reconstruction (ASR). The ancestral GXP evolved several times into the
same structure. The high degree of convergence was the first line of evidence that
GxpS evolution is driven by positive selection.
Chapter four aimed to analyze the driving forces for GxpS evolution. For that, I
examined every recombination event that has occurred in the evolution of GxpS. The
fact that I found a large number of synonymous (non-peptide-changing)
recombinations indicated that drift plays a significant role in the evolution of GxpS.
Further, I classified recombination into homotypic (recombination between A-domains
of equal specificity) and heterotypic recombination (recombination between A-domains
of unequal specificity). I found that heterotypic recombinations are significantly
different (longer and recombine more often in the specificity pocket) from homotypic
recombinations that I used as a proxy for neutral evolution in NRPS. That implied that
selection had acted at least in part on heterotypic recombinations. Next, I synthesized
a subset of evolutionary important GXP and tested them for insecticidal activity. The
bioactivity data revealed that the different structures of GXP have created species
specific compounds, which is another piece of evidence that selection has acted on
peptide diversity to overcome acquired resistance or to target new insect prey.
Simultaneously, a few structurally different peptides showed equal activity which again
suggests that there is an accessible sequence space that can be explored by drift to
create different but equally active peptides.
Overall, this works shows that NRPS evolution is rapid, creating a myriad of peptides
that can help in the battle against constantly developing resistance.
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