Genomics

Dataset Information

39

GAF zinc


ABSTRACT: 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 non-reproducible 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 survival of 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. Keywords: GAF zinc Overall design: Selection for genes essential for survival of high zinc conditions. Aliquots of a marinerT7 transposon library generated in S. pneumoniae R6 containing approximately 20,000 independent mutants were diluted 1 to 20 in GM17 broth or in GM17 broth containing 0.5 mM ZnSO4. Both cultures were grown for 10 generations as determined by OD600 after which the bacteria were spun down (9,000g, 4 min) and used for the extraction of genomic DNA.

INSTRUMENT(S): Streptococcus pneumoniae TIGR4/R6 v5 GAF

SUBMITTER: Anne de Jong  

PROVIDER: GSE6911 | GEO | 2007-06-26

SECONDARY ACCESSION(S): PRJNA98527

REPOSITORIES: GEO

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Publications

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

Bijlsma Jetta J E JJ   Burghout Peter P   Kloosterman Tomas G TG   Bootsma Hester J HJ   de Jong Anne A   Hermans Peter W M PW   Kuipers Oscar P OP  

Applied and environmental microbiology 20070119 5


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  ...[more]

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