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, 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.
Kraiss
A
A
Zentrum für Infektionsforschung, Universität Würzburg, 97070 Würzburg, Germany.
Schlör
S
S
Reidl
J
J
D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005838:Genotype; D006193:Haemophilus influenzae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012093:Replicon; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase