Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40–75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.
Integrative and conjugative elements (ICEs) are mobile genetic elements that can transfer by conjugation to recipient cells. Some ICEs integrate into a unique site in the genome of their hosts. We studied quantitatively the process by which an ICE searches for its unique integration site in the Bacillus subtilis chromosome. We followed the motion of both ICEBs1 and the chromosomal integration site in real time within individual cells. ICEBs1 exhibited a wide spectrum of dynamical behaviors, ranging from rapid sub-diffusive displacements crisscrossing the cell, to kinetically trapped states. The chromosomal integration site moved sub-diffusively and exhibited pronounced dynamical asymmetry between longitudinal and transversal motions, highlighting the role of chromosomal structure and the heterogeneity of the bacterial interior in the search. The successful search for and subsequent recombination into the integration site is a key step in the acquisition of integrating mobile genetic elements. Our findings provide new insights into intracellular transport processes involving large DNA molecules.
more » « less- NSF-PAR ID:
- 10396405
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 51
- Issue:
- 7
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- p. 3116-3129
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Mobile genetic elements (MGEs) are transient genetic material that can move either within a single organism's genome or between individuals or species. While historically considered “junk” DNA (i.e., deleterious or at best neutral), more recent studies reveal the potential adaptive advantages MGEs provide in lineages across the tree of life. Ciliates, a group of single‐celled microbial eukaryotes characterized by nuclear dimorphism, exemplify how epigenetic influences from MGEs shape genome architecture and patterns of molecular evolution. Ciliate nuclear dimorphism may have evolved as a response to transposon invasion and ciliates have since co‐opted transposons to carry out programmed DNA deletion. Another example of the effect of MGEs is in providing mechanisms for lateral gene transfer (LGT) from bacteria, which introduces genetic diversity and, in several cases, may drive ecological specialization in ciliates. As a third example, the integration of viral DNA, likely through transduction, provides new genetic materials and can change the way host cells defend themselves against other viral pathogens. We argue that the acquisition of MGEs through non‐Mendelian patterns of inheritance, coupled with their effects on ciliate genome architecture and persistence throughout evolutionary history, exemplify how the transmission of mobile elements should be considered a mechanism of transgenerational epigenetic inheritance.
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Conclusions Overall, our results suggest that the activated sludge process acted as a barrier against the proliferation of most ARGs, while those that persisted or increased warrant further attention.
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