Identification of Haemophilus influenzae Rd transformation genes using cassette mutagenesis

Abstract

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 mutants and comparison of genome sequence tags to genomic sequence data. The cassette mutagenesis screen for transformation genes resulted in five distinct mutant classes, two of which have been identified in previous studies. Insertions in the three newly identified loci interrupted genes with predicted protein products homologous to a type IV pilin-like protein biogenesis operon, drug-efflux transporters and a phospholipid-biosynthesis enzyme. The most significant finding of this screen is the requirement for type IV pilin-like proteins in genetic transformation of H. influenzae. These surface structures are utilized for DNA uptake in a number of Gram-positive and Gram-negative bacteria, and appear to be the common component among the systems for DNA binding.