Abstract
High-throughput sequencing of transposon (Tn) libraries created within entire genomes identifies and quantifies the contribution of individual genes and operons to the fitness of organisms in different environments. We used insertion-sequencing (INSeq) to analyze the contribution to fitness of all non-essential genes in the chromosome of Pseudomonas aeruginosa strain PA14 based on a library of ∼300,000 individual Tn insertions. In vitro growth in LB provided a baseline for comparison with the survival of the Tn insertion strains following 6 days of colonization of the murine gastrointestinal tract as well as a comparison with Tn-inserts subsequently able to systemically disseminate to the spleen following induction of neutropenia. Sequencing was performed following DNA extraction from the recovered bacteria, digestion with the MmeI restriction enzyme that hydrolyzes DNA 16 bp away from the end of the Tn insert, and fractionation into oligonucleotides of 1,200–1,500 bp that were prepared for high-throughput sequencing. Changes in frequency of Tn inserts into the P. aeruginosa genome were used to quantify in vivo fitness resulting from loss of a gene. 636 genes had <10 sequencing reads in LB, thus defined as unable to grow in this medium. During in vivo infection there were major losses of strains with Tn inserts in almost all known virulence factors, as well as respiration, energy utilization, ion pumps, nutritional genes and prophages. Many new candidates for virulence factors were also identified. There were consistent changes in the recovery of Tn inserts in genes within most operons and Tn insertions into some genes enhanced in vivo fitness. Strikingly, 90% of the non-essential genes were required for in vivo survival following systemic dissemination during neutropenia. These experiments resulted in the identification of the P. aeruginosa strain PA14 genes necessary for optimal survival in the mucosal and systemic environments of a mammalian host.
Highlights
The complex interaction of a pathogenic bacterium with a host leading to disease can be viewed as the coordinated and highlyregulated actions of a multitude of factors that allows the infecting organisms to successfully colonize tissue, occasionally disseminate and avoid the activities of host defense mechanisms
We generated a bank of approximately 300,000 Tn insertions in P. aeruginosa strain PA14 which was used to determine fitness dynamics in an infection cycle starting with colonization of the ceca followed by dissemination into the spleens after induction of neutropenia, as outlined in Figure 1 and described previously in detail [15]
To identify the genes that influence colonization, the ceca of the mice were harvested 6 days after colonization commenced (Figure 1A), the surviving P. aeruginosa strains with the Tn insertions grown, DNA extracted and digested with the MmeI enzyme, DNA fragments in the range of 1,200–1,500 bp obtained by gel fractionation and the recovered oligonucleotides prepared for high-throughput sequencing [6]
Summary
The complex interaction of a pathogenic bacterium with a host leading to disease can be viewed as the coordinated and highlyregulated actions of a multitude of factors that allows the infecting organisms to successfully colonize tissue, occasionally disseminate and avoid the activities of host defense mechanisms. DNA microarray based methods, such as signature-tagged mutagenesis [1,2,3] or transposon site hybridization [4,5], have been utilized to determine the importance of individual genes in the infection process based on the negative selection of mutants Such genomewide approaches provide another level of depth for understanding the role of virulence factors in the host, since they analyze the growth phenotype of individual bacterial mutants in the context of the entire population, i.e., in cells surrounded by otherwise phenotypically wild type (WT) siblings. The availability of high-throughput DNA sequencing technologies makes it feasible to obtain millions of DNA sequences from a single microbial sample This tool has emerged as a major means to detect variations in genetic fitness of individual mutants in a population undergoing selection in infected hosts. By preparing highly saturated random transposon (Tn) insertion libraries using a designed Tn followed by ascertaining the site of insertion via sequencing of the Tn junctions within the chromosomal DNA (variably referred to as Tn-seq or INSeq) [6,7,8], unique insights into the role of individual virulence factors and Author Summary
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