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Applied and Environmental Microbiology, December 1998, p. 4697-4702, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

In Vivo Transposon Mutagenesis in Haemophilus influenzae

Anita Kraiß, Stefan Schlör, and Joachim Reidl*

Zentrum für Infektionsforschung, Universität Würzburg, 97070 Würzburg, Germany

Received 8 June 1998/Accepted 22 September 1998

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, which are suitable for in vivo transposition in H. influenzae. The transposon delivery system was designed to contain an H. influenzae-specific uptake signal sequence which facilitates DNA transformation into H. influenzae. The following mini-Tn10 elements have been made suitable for specific tasks in H. influenzae: (i) Tn10d-cat, which can be used to generate chloramphenicol-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 transcriptional fusion system. For the rapid identification of the transposon insertions, a PCR fragment enrichment method was developed. This report demonstrates that this in vivo mutagenesis technique is a convenient tool for the analysis of biochemical and regulatory pathways in the human pathogen H. influenzae.

* Corresponding author. Mailing address: Zentrum für Infektionsforschung, Universität Würzburg, Röntgenring 11, 97070 Würzburg, Germany. Phone: 49-(0)931-312153. Fax: 49-(0)931-312578. E-mail: joachim.reidl{at}rzroe.uni-wuerzburg.de.

Applied and Environmental Microbiology, December 1998, p. 4697-4702, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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