Interplay between an integrative and conjugative element and its bacterial host

Horizontal gene transfer is the mechanism by which bacteria acquire new genes. Mobile genetic elements (MGEs) are the primary drivers of horizontal gene transfer, introducing large fragments of DNA. Often, MGEs contain cargo genes beneficial to the host, conferring new traits such as antibiotic resistances, pathogenicity, and metabolic capabilities. Though MGEs sometimes carry beneficial genes, they can impose fitness costs on their host. MGEs employ various maintenance and acquisition strategies to ensure stable acquisition in bacterial populations. Two common mechanisms are integration and replication. Integration and replication are inherently harmful, as they produce DNA intermediates recognized by the host as DNA damage. Thus, MGEs have evolved mechanisms to modulate, subvert, and manipulate bacterial DNA damage repair systems. Integrative and conjugative elements (ICEs) are considered the most abundant conjugative elements. ICEs are MGEs that reside integrated into their host chromosome, excise, and transfer their DNA via conjugation (involving a type IV secretion system) into a recipient. During acquisition, the element must switch from a replicating element to a quiescent element capable of integrating for stable acquisition. Here I describe for the model ICE, ICEBs1 of Bacillus subtilis, three mechanisms used to regulate and minimize the cost of autonomous replication for its bacterial host. I show how ICEBs1 couples the cessation of replication to integration into the chromosome of nascent hosts. I found that the integration of a replicating element is lethal for the host. I show that two genes encoded on ICEBs1 inhibit the host DNA damage repair response by preventing RecA filament formation. This activity confers a fitness advantage to the host in that it dampens the SOS response and protects against phage activation while also stabilizing ICEBs1 DNA. I also present preliminary evidence that ICEBs1 utilizes a host-specific DNA motif (Chi sites) that modulates the activity of an essential host exonuclease from degradative to reparative. I found that removing Chi sites on ICEBs1 resulted in decreased acquisition of the element in new hosts in mixed population biofilms. I also found evidence that ICEs of other organisms may use their host Chi sites. Together these findings show several mechanisms used by an integrative and conjugative element to maximize the spread and propagation of the element with minimal perturbations to its host.Ph.D

Randomly barcoded transposon mutant libraries for gut commensals II: Applying libraries for functional genetics

Summary: The critical role of the intestinal microbiota in human health and disease is well recognized. Nevertheless, there are still large gaps in our understanding of the functions and mechanisms encoded in the genomes of most members of the gut microbiota. Genome-scale libraries of transposon mutants are a powerful tool to help us address this gap. Recent advances in barcoded transposon mutagenesis have dramatically lowered the cost of mutant fitness determination in hundreds of in vitro and in vivo experimental conditions. In an accompanying review, we discuss recent advances and caveats for the construction of pooled and arrayed barcoded transposon mutant libraries in human gut commensals. In this review, we discuss how these libraries can be used across a wide range of applications, the technical aspects involved, and expectations for such screens