Integration host factor is required for replication of pYGK-derived plasmids in Aggregatibacter actinomycetemcomitans

Abstract

In this study, we show that integration host factor protein (IHF) is required for replication of pYGK 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 pYGK. We also identified two motifs that resemble the consensus IHF-binding site in a 813-bp fragment containing the pYGK 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 pJT1 were modified to generate plasmid pJT10. The introduction of these new unique sites in pJT10 facilitates the transfer of transcriptional or translational lacZ fusion constructs for the generation of single-copy chromosomal insertion of the reporter construct. Plasmid pJT10 and its derivatives will be useful for genetic studies in Aggregatibacter (Actinobacillus) and probably other genera of Pasteurellaceae, including Haemophilus, Pasteurella, and Mannheimia.

Keywords: Pasteurellaceae; integration host factor; plasmid replication.

Fig. 1

Binding of reconstituted integration host factor (IHF) heterodimer to the intergenic region (IGR) between orf1 and orf2 from pYGK plasmid. (a) Schematic representation of the 813-bp region from pYGK showing the putative binding regions for IHF (black circles), ORFs (open arrows) direct repeat regions: DR₁, DR₂ (same direction arrows), and invert repeat regions: IR₁, IR₂, IR₃, IR₄ (opposite direction arrows). PCR fragments used for EMSA reactions encompassing the orf1 and orf2 coding regions and the IGR sequence are numbered from I to XII and the nucleotides contained in each fragment are indicated to the right. The numbering of the nucleotides comprising the 813-bp sequence is relative to upstream region of the ORF1. For probes X and XII, M represents the site-specific mutations that were introduced into the putative IHF-binding sites. (b) PCR probes were incubated in the presence (+) or absence (−) of 4 μM reconstituted IHF heterodimer for 20 min at room temperature, and DNA–protein complexes were resolved in 6% polyacrylamide gels. A 200-bp DNA fragment from the psaA gene of Yersinia pseudotuberculosis (probe XIII) was used as the negative control.

Fig. 2

Double stranded DNA sequence of the orf1-orf2 IGR from pYGK plasmid. The numbers on the right hand side represent nucleotides upstream from the start codon of orf1 to the end codon of orf2. The start and end codons for orf1 and orf2 are indicated with a square arrow and are presented in boldface type. Regions that resemble the consensus IHF-binding site are underlined and labeled IHF₁ and IHF₂. Nucleotides of each of the putative IHF-binding sites that were altered by site-specific mutagenesis are shown above the DNA sequence.

Fig. 3

Physical maps of pYGK-derived and pJT1-derived plasmids. The cloning vector pJT7 (a), the transcriptional/translation lacZ reporter plasmid pJT9 (b), and the suicide vector pJT10 (c) are shown and were constructed as described in Materials and Methods. (d) Estimation of the copy number from pYGK-derived plasmids. The supercoiled plasmid DNA was obtained from Escherichia coli XL1-Blue MRF’ harboring individually pUC18 (high copy), pBR322 (low copy), pSC101 (very low copy), pYGK (low copy), pJT4, and pJT7. Each culture was grown in LB broth supplemented with the appropriate antibiotic [OD_{600 nm} of 0.6 (4 × 10⁷ colony-forming units, CFU, mL⁻¹)]. Supercoiled plasmid DNA was obtained from Aggregatibacter actinomycetemcomitans 652 strain harboring individually pYGK, pJT4, or pJT7. Cultures were grown in BHI broth supplemented with kanamycin [OD_{600 nm} of 0.6 (1.5 × 10⁶ CFU, mL⁻¹)]. The supercoiled plasmid DNA was separated by electrophoresis on a 1% agarose gel and stained with ethidium bromide. The figure is a negative image of a digital photograph of the ethidium bromide stained gel. The experiment was performed independently three times.