Comparison of transposon insertions in the mutant library before and after selection in the lung model. Insertion sites in the 5′ 5–80% protein coding sequence of the gene and reads associated with these sites were considered for fitness analysis. The saturation of transposon insertions within 1,239 genes in the input library is shown on the x axis. Saturation was calculated as the percentage of sites within a gene sustaining transposon insertions to the total number of possible of insertion sites (TA dinucleotides). The lung survival index (s.i.) is represented on the y axis as the number of reads mapped to a gene in the output library divided by the reads identified in the input library (points on the x axis represent s.i. values of zero). Essential genes, those sustaining insertions in <5% of possible sites, are not shown (shaded); the majority of these sustained no insertions, and the remaining 25% averaged 1 insertion per gene. The threshold for an inferred in vitro growth defect (solid line) was set at a saturation of 40% of the possible TA insertion sites within a gene. The threshold for in vivo attenuation (dashed line) was set at a lung s.i. of <0.30. Numbers of genes falling within each quadrant are indicated.

HITS and comparison of selected libraries. (A) HITS sample preparation and enrichment of transposon/chromosome junctions. After transposon mutagenesis, chromosomal DNA is purified from H. influenzae mutant library. Red, ITRs of himar1 transposon; white, contents of transposon, including the kanamycin resistance gene. Illumina oligonucleotide adapters (gray) are ligated to sheared genomic DNA. Fragments of the transposon/chromosome junctions are enriched via PCR using transposon- and adapter-specific primers. The biotinylated transposon-specific primer (yellow) anneals to the ITRs of the transposon and includes the Illumina sequencing primer site. The adapter-specific primer anneals to only 1 oligonucleotide of the partially complementary adapter. Enriched fragments are collected using streptavidin-coated paramagnetic beads. After washing, single-stranded DNAs are eluted from the beads and used for cluster formation on Illumina flow cells. (B) Comparison of lung-selected output library to input library. After sequencing, reads are mapped to the reference genome (solid arrows, plus strand; dashed arrows, minus strand) to identify the transposon insertion sites. The number of insertion sites detected per gene and the number of sequencing reads per site are used to determine the relative abundance of the mutant within the library before and after selection. The examples depict insertion patterns at TA sites in hypothetical gene A, in which insertion mutations confer attenuated growth or survival during infection, and gene B that is not required for growth in vitro or in vivo. Insertions in genes that are essential for growth on rich culture media are absent in the input library and are not detected by HITS.

Comparison of HITS analysis, genetic footprinting, and single-strain infections. Genetic footprinting of input and output libraries for (A) galU, encoding UDP-glucose pyrophosphorylase, (B) orfH, encoding heptosyltransferase III, and (C) xylA, encoding xylose isomerase, are shown in the gel images. PCR analysis was conducted using the transposon-specific primer marout and chromosomal primers galU_F, orfH_F, or xylA_F that anneal 278 bp, 202 bp, and 279 bp upstream of the respective genes. In the plots, HITS data correspond to regions analyzed by footprinting. (D) H. influenzae NTHi wild-type (NT127) and deletion mutants of galU and orfH recovered from lungs of C57BL/6 mice (7 mice per strain) 24 h after intranasal inoculation with each strain. Bars represent the mean cfu per lung. Comparisons between wild-type and mutants were statistically significant via one-way ANOVA with Tukey's multiple comparison test (P < 0.001). LLD, lower limit of detection. Fold differences in mean cfu recovered for NT127 wild-type strain vs. the galU or orfH mutants in individual mutant infections (brackets) are compared with HITS results (below the chart). HITS survival indices were 0.011 for galU and 0.012 for orfH, corresponding to in vivo attenuations (calculated as the reciprocal of the s.i.) of 89-fold and 82-fold, respectively. In A–C, genome coordinates of transposon insertion sites detected via HITS analysis were reoriented with respect to the chromosomal primer positions used in footprinting. The y axis was modeled to the migration of the molecular weight (MW) standard of footprinting gels using nonlinear regression, and the x axis represents the number of sequencing reads mapped to insertion sites. The scale of the MW standards on the right of each panel applies to both the genetic footprints and the HITS analysis plots. White, nonessential genes; gray, genes required for growth or survival in the lung.