Genome scanning in Haemophilus influenzae for identification of essential genes

We have developed a method for identifying essential genes by using an in v itro transposition system, with a small (975 bp) insertional element contai ning an antibiotic resistance cassette, and mapping these inserts relative to the deduced open reading frames of Haemophilus influenzae by PCR and Sou thern analysis. Putative essential genes are identified by two methods: mut ation exclusion or zero time analysis. Mutation exclusion consists of growi ng an insertional library and identifying open reading frames that do not c ontain insertional elements: in a growing population of bacteria, insertion s in essential genes are excluded. Zero time analysis consists of monitorin g the fate of individual insertions after transformation in a growing cultu re: the loss of inserts in essential genes is observed over time, Both meth ods of analysis permit the identification of genes required for bacterial s urvival. Details of the mutant library construction and the mapping strateg y, examples of mutant exclusion, and zero time analysis are presented.