Identification of Haemophilus influenzae Rd transformation genes using cassette mutagenesis

Genes required for natural transformation of Haemophilus influenzae Rd were identified by a cassette mutagenesis protocol consisting of the following steps: random insertional mutagenesis, phenotypic screening, sequencing of genome sequence tags from the DNA flanking the insertion in the selected mu tants and comparison of genome sequence tags to genomic sequence data. The cassette mutagenesis screen for transformation genes resulted in five disti nct mutant classes, two of which have been identified in previous studies. Insertions in the three newly identified loci interrupted genes with predic ted protein products homologous to a type IV pilin-like protein biogenesis operon, drug-efflux transporters and a phospholipid-biosynthesis enzyme. Th e most significant finding of this screen is the requirement for type IV pi lin-like proteins in genetic transformation of H. influenzae. These surface structures are utilized for DNA uptake in a number of Gram-positive and Gr am-negative bacteria, and appear to be the common component among the syste ms for DNA binding.