Uncovering the rules governing mobile genetic element spread

Horizontal gene transfer plays a major role in the evolution of microbes and host-microbe interactions. The key drivers of horizontal gene transfer are mobile genetic elements (MGEs), diverse DNA elements that mobilize genes directly between and within cells in all branches of life. MGEs, such as plasmids and integrative and conjugative elements (ICEs) of bacteria, are implicated in the spread of genes important for pathogenicity, symbiosis, antimicrobial resistance (AMR), and other adaptive functions. While the role of MGEs in many host- microbe interactions is well known, the factors promoting or constraining MGE spread and microbial host compatibility are poorly understood. This proposal aims to characterize MGE compatibility at multiple scales, from interactions between individual MGEs and strains, among all strains and MGEs of a genus, to interactions in complex microbial communities. We will use plasmids of the well-studied agrobacteria/rhizobia complex as a model to uncover generalizable concepts on the evolution of plasmid-bacteria-host interactions. Several features make this system advantageous, including genetic tractability, the ability to control microbiome composition, and experiments with large sample size. First, we will characterize plasmid incompatibility by investigating the diversity of replication and transfer loci on all sequenced plasmids of a genus. We will characterize other barriers to MGE transfer by investigating variation in restriction-modification systems, DNA methylation, and defense/anti- defense systems across this genus-level group. We will then overlay this information and build a model to predict, for every plasmid, its compatibility with a given recipient strain. Second, we will investigate how MGEs and chromosomes co-evolve to maintain compatibility. We will focus on a family of plasmids restricted to polyphyletic host lineages and extend this model to predict other plasmid or chromosomal loci necessary for compatibility with these MGEs. We will use both genome reduction (Tn-Seq) and additive (cosmid library) screens to identify loci determining compatibility. Third, we will investigate factors determining the extent and frequency of MGE transfer in complex microbial communities. Long-read, methylation-aware metagenomics will inform on the diversity of MGEs in the disease environment. Hi-C sequencing and/or single-cell fusion PCR will be used to determine the association of MGEs with specific microbials hosts. The presence of restriction- modification systems, DNA methylation, and defense systems in members of these communities will inform on constraints of MGE transfer. Overall, this work will provide a holistic model of constraints on MGE spread and will shed light on how MGEs and their hosts co-evolve to maintain compatibility. Project Number: 1R35GM160469-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Alexandra Weisberg | Institution: OREGON STATE UNIVERSITY, CORVALLIS, OR | Award Amount: $408,375 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award A Study Section[MRAA] View on NIH RePORTER: https://reporter.nih.gov/project-details/11202743