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Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung.

ABSTRACT: Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale "negative selection" technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model. Mutations in 136 genes resulted in defects in vivo, and quantitative estimates of fitness generated by this technique were in agreement with independent validation experiments using individual mutant strains. Genes required in the lung included those with characterized functions in other models of H. influenzae pathogenesis and genes not previously implicated in infection. Genes implicated in vivo have reported or potential roles in survival during nutrient limitation, oxidative stress, and exposure to antimicrobial membrane perturbations, suggesting that these conditions are encountered by H. influenzae during pulmonary infection. The results demonstrate an efficient means to identify genes required for bacterial survival in experimental models of pathogenesis, and this approach should function similarly well in selections conducted in vitro and in vivo with any organism amenable to insertional mutagenesis.

SUBMITTER: Gawronski JD

PROVIDER: S-EPMC2752563 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung.

Gawronski Jeffrey D JD Wong Sandy M S SM Giannoukos Georgia G Ward Doyle V DV Akerley Brian J BJ

Proceedings of the National Academy of Sciences of the United States of America 20090904 38

Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale "negative selection" technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to d ...[more]

PMID: 19805314

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Project description:Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a 'phenotype of interest' (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with 'Fuzzy-Spreader'-like morphologies were also identified through a visual screen. The second, a 'mutant of interest' (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid.

Dataset Information

Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung.

Publications

Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung.

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