Integration host factor is required for replication of p YGK ‐derived plasmids in A ggregatibacter actinomycetemcomitans

In this study, we show that integration host factor protein (IHF) is required for replication of p YGK plasmids in Aggregatibacter actinomycetemcomitans . YGK plasmids were not replicated in A. actinomycetemcomitans strains lacking either the α‐ or β‐ subunit of IHF. However, the deletion mutants were complemented, and plasmid replication was restored when the promoter region and gene for either ihfA or ihfB was cloned into p YGK . We also identified two motifs that resemble the consensus IHF‐binding site in a 813‐bp fragment containing the p YGK origin of replication. Using electrophoretic mobility shift assays, purified IHFα–IHFβ protein complex was shown to bind to probes containing either of these motifs. To our knowledge, this is the first report showing that plasmid replication is IHF‐dependent in the family Pasteurellaceae . In addition, using site‐direct mutagenesis, the XbaI and KpnI restriction sites in the suicide vector p JT 1 were modified to generate plasmid p JT 10. The introduction of these new unique sites in p JT 10 facilitates the transfer of transcriptional or translational lacZ fusion constructs for the generation of single‐copy chromosomal insertion of the reporter construct. Plasmid p JT 10 and its derivatives will be useful for genetic studies in Aggregatibacter ( Actinobacillus ) and probably other genera of Pasteurellaceae , including Haemophilus , Pasteurella, and Mannheimia .