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Journal of Bacteriology, March 2001, p. 1694-1706, Vol. 183, No. 5
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.5.1694-1706.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Genetic Footprinting in Bacteria†

Roberta S. Hare,1 Scott S. Walker,1,* Thomas E. Dorman,2 Jonathan R. Greene,1 Luz-Maria Guzman,2,Dagger Teresa J. Kenney,2 Mark C. Sulavik,2 Khandan Baradaran,2 Chad Houseweart,2 Haiying Yu,2 Zuzana Foldes,2 Anna Motzer,2 Michael Walbridge,2 George H. Shimer Jr.,2 and Karen Joy Shaw1

Schering-Plough Research Institute, Kenilworth, New Jersey 07033,1 and Genome Therapeutics Corporation, Waltham, Massachusetts 024532

Received 22 September 2000/Accepted 7 December 2000

In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage lambda  transposon delivery system.

* Corresponding author. Mailing address: Schering-Plough Research Institute, 2015 Galloping Hill Road (4700), Kenilworth, NJ 07033-0539. Phone: (908) 740-7597. Fax: (908) 740-3918. E-mail: scott.walker{at}spcorp.com.

dagger This article is dedicated to the memory of Claire M. Berg, who advised us on the early stages of this project and was a dear mentor and friend.

Dagger Present address: Millennium Pharmaceuticals, Inc., 75 Sidney St., Cambridge, MA 02139.

Journal of Bacteriology, March 2001, p. 1694-1706, Vol. 183, No. 5
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.5.1694-1706.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.