Logo image
Back
Testing the limits of natural transformation: effects of donor DNA size and genomic structural variation on Haemophilus influenzae recombination
Dissertation   Open access

Testing the limits of natural transformation: effects of donor DNA size and genomic structural variation on Haemophilus influenzae recombination

Danielle Renee Piazza
Doctor of Philosophy (Ph.D.), Drexel University
Nov 2021
DOI:
https://doi.org/10.17918/00000926
pdf
Piazza_Danielle_202114.35 MBDownloadView
PDF Open Access Open Access (License Unspecified)

Abstract

Haemophilus influenzae Bacteria
Naturally transformable bacteria actively take up intact DNA molecules from their environment, which can be incorporated into their own chromosomes via homologous recombination. Analogous to sex in eukaryotes, natural transformation allows bacteria to rapidly adapt to new environments by acquiring traits from their dead relatives - for example, hastening evasion of host immunity or spreading antibiotic resistances. Although mechanistic biases are known to arise during meiotic recombination in sexual eukaryotes, little is known about such biases during bacterial natural transformation, nor how they affect pathogen evolution. Here, we used Haemophilus influenzae as a model to investigate the genome-scale consequences of natural transformation by varying the size and divergence of taken up donor DNAs. Deep sequencing reveals >100-fold variation in transformation rates at different loci when donor DNAs were short, and this effect was partly explained by local sequence similarity between donor and recipient. Identified hotspot and coldspots of transformation are also present in natural populations, and were validated by an allele-specific droplet digital PCR assay (ddPCR), developed as an independent tool to measure these loci with more flexibility, reproducibility, and cost effectiveness compared to deep sequencing efforts. We also found that single cells could take up and recombine exceptionally long DNAs spanning >10% of chromosome length. Surprisingly, co-transformation was mostly unimpaired across inversions and other rearrangements, though the breakpoints of such structural variants often acted as barriers. We also present novel applications of DNA optical mapping, which could disentangle transformation itself from its consequences on fitness. Genetic incompatibilities, which result in altered fitness of the recombinants, require addressing to fully understand the consequences of genetic divergence or marker selection on fitness. We developed methods to chemically label donor DNA, transform it into recipient chromosomes, and then use OM to detect individual transformation events at a single-molecule level with extremely high physical coverage (>100K-fold). In the future, this will allow us to make genome-scale transformation maps while minimizing fitness effects of recombination and without relying on genetic variation. Overall, our results show that H. influenzae can take up and recombine much larger molecules than previously expected and underline the importance of bacterial recombination in the evolution of pathogenesis. These findings have expanded the field of sequencing of bacterial recombinants and provide new opportunities to study population level transformation biases. This work suggests new mechanistic investigations, will inform genomic epidemiology studies, and advances the search for novel vaccine and drug targets.

Metrics

59 File views/ downloads
23 Record Views

Details

Logo image