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.

A Primed Subpopulation of Bacteria Enables Rapid Expression of the Type 3 Secretion System in Pseudomonas aeruginosa.

Type 3 secretion systems (T3SS) are complex nanomachines that span the cell envelope and play a central role in the biology of Gram-negative pathogens and symbionts. In Pseudomonas aeruginosa, T3SS expression is strongly associated with human disease severity and with mortality in murine acute pneumonia models. Uniform exposure of isogenic cells to T3SS-activating signal results in heterogeneous expression of this critical virulence trait. To understand the function of such diversity, we measured the production of the T3SS master regulator ExsA and the expression of T3SS genes using fluorescent reporters. We found that heterogeneous expression of ExsA in the absence of activating signal generates a "primed" subpopulation of cells that can rapidly induce T3SS gene expression in response to signal. T3SS expression is accompanied by a reproductive trade-off as measured by increased division time of T3SS-expressing cells. Although T3SS-primed cells are a minority of the population, they compose the majority of T3SS-expressing cells for several hours following activation. The primed state therefore allows P. aeruginosa to maximize reproductive fitness while maintaining the capacity to quickly express the T3SS. As T3SS effectors can serve as shared public goods for nonproducing cells, this division of labor benefits the population as a whole. IMPORTANCE The expression of specific virulence traits is strongly associated with Pseudomonas aeruginosa's success in establishing acute infections but is thought to carry a cost for bacteria. Producing multiprotein secretion systems or motility organelles is metabolically expensive and can target a cell for recognition by innate immune system receptors that recognize structural components of the type 3 secretion system (T3SS) or flagellum. These acute virulence factors are also negatively selected when P. aeruginosa establishes chronic infections in the lung. We demonstrate a regulatory mechanism by which only a minority subpopulation of genetically identical P. aeruginosa cells is "primed" to respond to signals that turn on T3SS expression. This phenotypic heterogeneity allows the population to maximize the benefit of rapid T3SS effector production while maintaining a rapidly growing and nonexpressing reservoir of cells that perpetuates this genotype within the population.

Cheating by type 3 secretion system-negative Pseudomonas aeruginosa during pulmonary infection.

The opportunistic pathogen Pseudomonas aeruginosa expresses a type 3 secretion system (T3SS) strongly associated with bacterial virulence in murine models and human patients. T3SS effectors target host innate immune mechanisms, and T3SS-defective mutants are cleared more efficiently than T3SS-positive bacteria by an immunocompetent host. Nonetheless, T3SS-negative isolates are recovered from many patients with documented P. aeruginosa infections, leading us to test whether T3SS-negative strains could have a selective advantage during in vivo infection. Mice were infected with mixtures of T3SS-positive WT P. aeruginosa plus isogenic T3SS-OFF or constitutively T3SS-ON mutants. Relative fitness of bacteria in this acute pneumonia model was reflected by the competitive index of mutants relative to WT. T3SS-OFF strains outcompeted WT PA103 in vivo, whereas a T3SS-ON mutant showed decreased fitness compared with WT. In vitro growth rates of WT and T3SS-OFF bacteria were determined under T3SS-inducing conditions and did not differ significantly. Increased fitness of T3SS-OFF bacteria was no longer observed at high ratios of T3SS-OFF to WT, a feature characteristic of bacterial cheaters. Cheating by T3SS-OFF bacteria occurred only when T3SS-positive bacteria expressed the phospholipase A2 effector Exotoxin U (ExoU). T3SS-OFF bacteria showed no fitness advantage in competition experiments carried out in immunodeficient MyD88-knockout mice or in neutrophil-depleted animals. Our findings indicate that T3SS-negative isolates benefit from the public good provided by ExoU-mediated killing of recruited innate immune cells. Whether this transient increase in fitness observed for T3SS-negative strains in mice contributes to the observed persistence of T3SS-negative isolates in humans is of ongoing interest.