In vivo transposon mutagenesis in Haemophilus influenzae

In order to devise an in vivo insertion mutagenesis scheme for Haemophilus influenzae, a novel set of transposons has been constructed. These are Tn10 -based minitransposons carried on pACYC184- and pACYC177-based replicons, w hich are suitable for in vivo transposition in H. influenzae. The transposo n delivery system was designed to contain an H, influenzae-specific uptake signal sequence which facilitates DNA transformation into H. influenzae. Th e following mini-Tn10 elements have been made suitable for specific tasks i n H, influenzae: (i) Tn10d-cat, which can be used to generate chloramphenic ol-selectable insertion mutations; (ii) Tn10d-bla, an ampicillin-selectable translational fusion system allowing the detection of membrane or secreted proteins; and (iii) Tn10d-lacZcat, a chloramphenicol-selectable lacZ trans criptional fusion system. For the rapid identification of the transposon in sertions, a PCR fragment enrichment method was developed. This report demon strates that this in vivo mutagenesis technique is a convenient tool for th e analysis of biochemical and regulatory pathways in the human pathogen H, influenzae.