CONSTRUCTION OF ANTIBIOTIC-RESISTANCE CASSETTES WITH MULTIPLE PAIRED RESTRICTION SITES FOR INSERTIONAL MUTAGENESIS OF HAEMOPHILUS-INFLUENZAE

Insertional mutagenesis of cloned genes coupled with site specific rec ombination into the genome of the parent organism is an ideal method f or characterizing gene function. In this paper we describe the product ion and utility of two antibiotic resistance cassettes for use in Haem ophilus influenzae. The mutagenic elements encode resistance to chlora mphenicol or spectinomycin. Multiple paired restriction enzyme sites b ound both cassettes. Use of these constructs to create mutants in H. i nfluenzae demonstrated that the cassettes are readily incorporated int o the genome in single copy and allow easy detection of mutant constru cts. The insertions are stable following repeated in vitro passage. In addition, the elements are compatible with each other and allow the c onstruction of multiple mutations within a single strain. (C) 1998 Pub lished by Elsevier Science B.V. All rights reserved.