Development of genomic array footprinting for identification of conditionally essential genes in Streptococcus pneumoniae.

Fulltext:
51900.pdf
Embargo:
until further notice
Size:
652.8Kb
Format:
PDF
Description:
publisher's version
Publication year
2007Source
Applied and Environmental Microbiology, 73, 5, (2007), pp. 1514-1524ISSN
Publication type
Article / Letter to editor

Display more detailsDisplay less details
Organization
Paediatrics
Biochemistry (UMC)
Journal title
Applied and Environmental Microbiology
Volume
vol. 73
Issue
iss. 5
Page start
p. 1514
Page end
p. 1524
Subject
N4i 1: Pathogenesis and modulation of inflammation; N4i 4: Auto-immunity, transplantation and immunotherapy; NCMLS 1: Infection and autoimmunity; UMCN 4.1: Microbial pathogenesis and host defenseAbstract
Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput, genome-wide technique, genomic array footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in nonreproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1 showed that both have an equal degree of saturation but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for surviving zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high-zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae.
This item appears in the following Collection(s)
- Academic publications [204859]
- Electronic publications [103204]
- Faculty of Medical Sciences [81031]
Upload full text
Use your RU credentials (u/z-number and password) to log in with SURFconext to upload a file for processing by the repository team.