Random Chromosomal Gene Disruption Using Cassette Mutagenesis

Random mutagenesis of DNA has been an essential tool for investigation of bacteria, fungi, and other organisms. Given the recent explosion in genomic sequence information (e.g., over the past 5 yr there have been 30 published microbial genomes and over 100 microbial genome sequencing projects), there is a growing need for reliable methods of random DNA mutagenesis to allow for functional genomics work. The authors were studying Haemophilus influenzae , the first organism to have its genome completely sequenced (1 ). H. influenzae is a Gram-negative facultative anaerobe, which resides in the human upper respiratory tract and causes disease. H. influenzae is also capable of taking DNA from its environment and integrating it into the bacterial chromosome (2 ,3 ). A number of genes required for DNA transformation have been identified in H. influenzae. These studies have used a variety of molecular methods to identify transformation genes, including complementation of transformation-deficient mutants derived by chemical mutagenesis (4 ), a “poison-DNA” selection method (5 ), mini-transposon (Tn)10 mutagenesis (6 ), Tn916 mutagenesis (7 ), and other genetic selection techniques (8 ). In order to identify the remaining transformation genes by insertional mutagenesis, while overcoming potential biases inherent in Tn-based screens (e.g., hot-spotting), the authors decided to investigate the utility of the cassette mutagenesis protocol (9 ). In this procedure, an antibiotic-resistance cassette is ligated to restriction endonuclease-digested chromosome DNA, which has been treated so that the original gene order is maintained during the process. The resulting cassette-mutagenized DNA is then transformed into competent bacteria, resulting in the insertion of the cassette marker throughout the chromosome and the generation of mutants by gene disruption.