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. 2002 Jan 22;99(2):966-71.
doi: 10.1073/pnas.012602299.

A genome-scale analysis for identification of genes required for growth or survival of Haemophilus influenzae

Brian J Akerley  1 , Eric J Rubin, Veronica L Novick, Kensey Amaya, Nicholas Judson, John J Mekalanos
Affiliations

A genome-scale analysis for identification of genes required for growth or survival of Haemophilus influenzae

Brian J Akerley et al. Proc Natl Acad Sci U S A. .

Abstract

A high-density transposon mutagenesis strategy was applied to the Haemophilus influenzae genome to identify genes required for growth or viability. This analysis detected putative essential roles for the products of 259 ORFs of unknown function. Comparisons between complete genomes defined a subset of these proteins in H. influenzae having homologs in Mycobacterium tuberculosis that are absent in Saccharomyces cerevisiae, a distribution pattern that favors their use in development of antimicrobial therapeutics. Three genes within this set are essential for viability in other bacteria. Interfacing the set of essential gene products in H. influenzae with the distribution of homologs in other microorganisms can detect components of unrecognized cellular pathways essential in diverse bacteria. This genome-scale phenotypic analysis identifies potential roles for a large set of genes of unknown function.

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Figures

Figure 1
Figure 1
Strategy for whole-genome mutagenesis by in vitro mariner mutagenesis and functional analysis by genetic footprinting. (A) In vitro transposon mutagenesis with Tnmagellan4 conducted on PCR products corresponding to regions of the H. influenzae chromosome. Each mutagenized DNA segment was introduced separately into H. influenzae by natural transformation and homologous recombination. The Tnmagellan4 element encodes kanamycin resistance (KmR) and contains a DNA US, both of which are flanked by transposon-inverted repeats (black triangles). (B) PCR products (10 kb) were generated as targets for mutagenesis with ≈5 kb of overlap between adjacent segments. These products were mutagenized (as in A) to generate 366 individual pools of transposon mutants. The same primers were used to obtain genetic footprinting data for every ≈2.5 kb of the genome, and each primer was used on two independent pools of mutants.
Figure 2
Figure 2
Scoring system for genetic footprinting of the H. influenzae genome. Maps of ORF locations with respect to binding sites of each primer were superimposed on gel images. Dashed (putative nonessential regions) or solid (putative essential regions) lines on the gel image represent predicted segments of H. influenzae coding sequences. Primer reference numbers (red) are listed near primer positions on the gene map (Left) and on the gel image at the bottom of each lane. Tick marks on the gene map represent approximate positions where insertions were detected. Pools of mutants derived from independent transposition reactions are named by the primer pairs used to generate targets for in vitro transposon mutagenesis (top, brackets). The gel image shows analysis of two overlapping ≈10-kb regions corresponding to mutant pools 327–328 and 329–330. Colors denote the summarized results. Red , no insertions; blue, one insertion site; white, insertion mutations partially reduce growth; and yellow, evenly distributed insertions/putative nonessential gene. Absence of insertions within 300 bp of the end of a target PCR product was not considered significant because of the minimum distance required for homologous recombination (e.g., blue line marked with asterisk in lane 327).
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