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Journal of Bacteriology, August 2004, p. 4921-4930, Vol. 186, No. 15
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.15.4921-4930.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Systematic Mutagenesis of the Escherichia coli Genome{dagger}

Yisheng Kang,1,{ddagger} Tim Durfee,1*,{ddagger} Jeremy D. Glasner,2 Yu Qiu,1 David Frisch,1 Kelly M. Winterberg,3 and Frederick R. Blattner1

Department of Genetics,1 Department of Animal Health and Biomedical Sciences,2 Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 537063

Received 9 December 2003/ Accepted 14 April 2004

A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the {lambda}Red proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition.

* Corresponding author. Mailing address: Department of Genetics, University of Madison-Wisconsin, Rm. 206, 445 Henry Mall, Madison, WI 53706. Phone: (608) 262-2534. Fax: (608) 263-7459. E-mail: durf{at}genome.wisc.edu.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} Y.K. and T.D. contributed equally to this study.

Journal of Bacteriology, August 2004, p. 4921-4930, Vol. 186, No. 15
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.15.4921-4930.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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