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Applied and Environmental Microbiology, January 2005, p. 451-459, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.451-459.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Phenotypic Screening of Escherichia coli K-12 Tn5 Insertion Libraries, Using Whole-Genome Oligonucleotide Microarrays

Kelly M. Winterberg,1 John Luecke,2 Amanda S. Bruegl,3 and William S. Reznikoff1*

Department of Biochemistry,1 Genome Expression Center, University of Wisconsin—Madison, Madison, Wisconsin,2 University of Washington—Seattle, Seattle, Washington3

Received 21 May 2004/ Accepted 9 August 2004

Complete genome sequences in combination with global screening methods allow parallel analysis of multiple mutant loci to determine the requirement for specific genes in different environments. In this paper we describe a high-definition microarray approach for investigating the growth effects of Tn5 insertions in Escherichia coli K-12. Libraries of insertion mutants generated by a unique Tn5 mutagenesis system were grown competitively in defined media. Biotin-labeled runoff RNA transcripts were generated in vitro from transposon insertions in each population of mutants. These transcripts were then hybridized to custom-designed oligonucleotide microarrays to detect the presence of each mutant in the population. By using this approach, the signal associated with 25 auxotrophic insertions in a 50-mutant pool was not detectable following nine generations of growth in glucose M9 minimal medium. It was found that individual insertion sites could be mapped to within 50 bp of their genomic locations, and 340 dispensable regions in the E. coli chromosome were identified. Tn5 insertions were detected in 15 genes for which no previous insertions have been reported. Other applications of this method are discussed.

* Corresponding author. Mailing address: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr., Room 319, Madison, WI 53706-1544. Phone: (608) 262-3608. Fax: (608) 265-2603. E-mail: reznikoff{at}biochem.wisc.edu.

Applied and Environmental Microbiology, January 2005, p. 451-459, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.451-459.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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