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Journal of Bacteriology, August 1999, p. 4961-4968, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Genome Scanning in Haemophilus influenzae for Identification of Essential Genes

Karl A. Reich,* Linda Chovan, and Paul Hessler

Genomics and Molecular Biology, Abbott Laboratories, Abbott Park, Illinois 60064

Received 3 November 1998/Accepted 10 June 1999

We have developed a method for identifying essential genes by using an in vitro transposition system, with a small (975 bp) insertional element containing an antibiotic resistance cassette, and mapping these inserts relative to the deduced open reading frames of Haemophilus influenzae by PCR and Southern analysis. Putative essential genes are identified by two methods: mutation exclusion or zero time analysis. Mutation exclusion consists of growing an insertional library and identifying open reading frames that do not contain insertional elements: in a growing population of bacteria, insertions in essential genes are excluded. Zero time analysis consists of monitoring the fate of individual insertions after transformation in a growing culture: the loss of inserts in essential genes is observed over time. Both methods of analysis permit the identification of genes required for bacterial survival. Details of the mutant library construction and the mapping strategy, examples of mutant exclusion, and zero time analysis are presented.

* Corresponding author. Mailing address: Abbott Laboratories, Dept. 4MD, Bldg. AP52-1N, 200 Abbott Park Rd., Abbott Park, IL 60064-6217. Phone: (847) 938-2635. Fax: (847) 938-3403. E-mail: karl.reich{at}abbott.com.

Journal of Bacteriology, August 1999, p. 4961-4968, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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