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Applied and Environmental Microbiology, May 1999, p. 1883-1890, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Construction and Analysis of a Library for Random Insertional Mutagenesis in Streptococcus pneumoniae: Use for Recovery of Mutants Defective in Genetic Transformation and for Identification of Essential Genes

Myeong S. Lee,1 Brian A. Dougherty,2,dagger Anne C. Madeo,1,Dagger and Donald A. Morrison1,*

Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois,1 and The Institute for Genomic Research, Rockville, Maryland2

Received 19 January 1999/Accepted 3 March 1999

To explore the use of insertion-duplication mutagenesis (IDM) as a random gene disruption mutagenesis tool for genomic analysis of Streptococcus pneumoniae, a large mutagenic library of chimeric plasmids with 300-bp inserts was constructed. The library was large enough to produce 60,000 independent plasmid clones in Escherichia coli. Sequencing of a random sample of 84 of these clones showed that 85% of the plasmids had inserts which were scattered widely over the genome; 80% of these plasmids had 240- to 360-bp inserts, and 60% of the inserts targeted internal regions of apparent open reading frames. Thus, the library was both complex and highly mutagenic. To evaluate the randomness of mutagenesis during recombination and to test the usefulness of the library for obtaining specific classes of nonessential genes, this library was used to seek competence-related genes by constructing a large pneumococcal transformant library derived from 20,000 mutagenic plasmids. After we screened the mutants exhaustively for transformation defects, 114 competence-related insertion mutations were identified. These competence mutations hit most previously known genes required for transformation as well as a new gene with high similarity to the Bacillus subtilis competence gene comFA. Mapping of the mutation sites at these competence loci showed that the mutagenesis was highly random, with no apparent hot spots. The recovery of a high proportion of competence genes and the absence of hot spots for mutational hits together show that such a transformant library is useful for finding various types of nonessential genes throughout the genome. Since a promoterless lacZ reporter vector was used for the construction of the mutagenic plasmid library, it also serves as a random transcriptional fusion library. Finally, use of a valuable feature of IDM, directed gene targeting, also showed that essential genes, which can be targets for new drug designs, could be identified by simple sequencing and transformation reactions. We estimate that the IDM library used in this study could readily achieve about 90% genome coverage.

* Corresponding author. Mailing address: Laboratory for Molecular Biology, Room 4110 MBR, 900 South Ashland Ave., Chicago, IL 60607. Phone: (312) 996-6839. Fax: (312) 413-2691. E-mail: DAMorris{at}uic.edu.

dagger Present address: Bristol-Myers Squibb PRI, Wallingford, CT 06492.

Dagger Present address: Department of Human Genetics, University of Michigan, Ann arbor, MI 48109.

Applied and Environmental Microbiology, May 1999, p. 1883-1890, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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