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

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.

Timing of integration into the chromosome is critical for the fitness of an integrative and conjugative element and its bacterial host.

Integrative and conjugative elements (ICEs) are major contributors to genome plasticity in bacteria. ICEs reside integrated in the chromosome of a host bacterium and are passively propagated during chromosome replication and cell division. When activated, ICEs excise from the chromosome and may be transferred through the ICE-encoded conjugation machinery into a recipient cell. Integration into the chromosome of the new host generates a stable transconjugant. Although integration into the chromosome of a new host is critical for the stable acquisition of ICEs, few studies have directly investigated the molecular events that occur in recipient cells during generation of a stable transconjugant. We found that integration of ICEBs1, an ICE of Bacillus subtilis, occurred several generations after initial transfer to a new host. Premature integration in new hosts led to cell death and hence decreased fitness of the ICE and transconjugants. Host lethality due to premature integration was caused by rolling circle replication that initiated in the integrated ICEBs1 and extended into the host chromosome, resulting in catastrophic genome instability. Our results demonstrate that the timing of integration of an ICE is linked to cessation of autonomous replication of the ICE, and that perturbing this linkage leads to a decrease in ICE and host fitness due to a loss of viability of transconjugants. Linking integration to cessation of autonomous replication appears to be a conserved regulatory scheme for mobile genetic elements that both replicate and integrate into the chromosome of their host.

Target search by an imported conjugative DNA element for a unique integration site along a bacterial chromosome during horizontal gene transfer.

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.