Improved plasmid shuttle vectors for Haemophilus influenzae and Escherichia coli

In vitro deletion and transposon mutagenesis experiments were performed to localize the region essential for plasmid RSF0885 replication to a 1.7-kb sequence downstream from the beta-lactamase gene. This locus was named rep, for replication. Plasmid RSF0885 can replicate in both Haemophilus influenzae and Escherichia coli. Replication in E. coli depended on transcription run-off from the beta-lactamase promoter into the rep locus. Insufficient transcription into the rep locus could account for the instability of this plasmid in an E. coli background, a property which reduced its usefulness as a shuttle vector. Therefore, four improved shuttle vectors for H. influenzae and E. coli were constructed. They possess the Co1E1 replication origin for maintenance in E. coli and the plasmid RSF0885 rep locus for maintenance in H. influenzae. Together, they provide twelve unique restriction sites for cloning by insertional inactivation of drug-resistance genes.