PMID- 17261526
OWN - NLM
STAT- MEDLINE
DCOM- 20070410
LR  - 20191210
IS  - 0099-2240 (Print)
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Linking)
VI  - 73
IP  - 5
DP  - 2007 Mar
TI  - Development of genomic array footprinting for identification of conditionally 
      essential genes in Streptococcus pneumoniae.
PG  - 1514-24
AB  - 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.
FAU - Bijlsma, Jetta J E
AU  - Bijlsma JJ
AD  - Department of Molecular Genetics, University of Groningen, Haren, The 
      Netherlands.
FAU - Burghout, Peter
AU  - Burghout P
FAU - Kloosterman, Tomas G
AU  - Kloosterman TG
FAU - Bootsma, Hester J
AU  - Bootsma HJ
FAU - de Jong, Anne
AU  - de Jong A
FAU - Hermans, Peter W M
AU  - Hermans PW
FAU - Kuipers, Oscar P
AU  - Kuipers OP
LA  - eng
SI  - GEO/GSM158348
SI  - GEO/GSM158350
SI  - GEO/GSM158558
SI  - GEO/GSM158562
SI  - GEO/GSM158563
SI  - GEO/GSM158564
SI  - GEO/GSM158565
SI  - GEO/GSM158566
SI  - GEO/GSM158567
SI  - GEO/GSM158568
SI  - GEO/GSM158569
SI  - GEO/GSM158570
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20070119
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (Bacterial Proteins)
RN  - 0 (DNA Transposable Elements)
RN  - J41CSQ7QDS (Zinc)
SB  - IM
MH  - Bacterial Proteins/genetics/metabolism
MH  - DNA Footprinting/*methods
MH  - DNA Transposable Elements/*genetics
MH  - Gene Expression Regulation, Bacterial
MH  - Gene Library
MH  - *Genes, Essential
MH  - *Genome, Bacterial
MH  - Heat-Shock Response
MH  - Humans
MH  - Molecular Sequence Data
MH  - Mutagenesis, Insertional
MH  - Oligonucleotide Array Sequence Analysis/*methods
MH  - Streptococcus pneumoniae/drug effects/*genetics/growth & development
MH  - Zinc/pharmacology
PMC - PMC1828782
EDAT- 2007/01/31 09:00
MHDA- 2007/04/11 09:00
PMCR- 2007/07/01
CRDT- 2007/01/31 09:00
PHST- 2007/01/31 09:00 [pubmed]
PHST- 2007/04/11 09:00 [medline]
PHST- 2007/01/31 09:00 [entrez]
PHST- 2007/07/01 00:00 [pmc-release]
AID - AEM.01900-06 [pii]
AID - 1900-06 [pii]
AID - 10.1128/AEM.01900-06 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 2007 Mar;73(5):1514-24. doi: 10.1128/AEM.01900-06. Epub 
      2007 Jan 19.