tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

Abstract

tfoX (sxy) is a regulatory gene needed to turn on competence genes. Aggregatibacter (Actinobacillus) actinomycetemcomitans has a tfoX gene that is important for transformation. We cloned this gene on an IncQ plasmid downstream of the inducible tac promoter. When this plasmid was resident in cells of A. actinomycetemcomitans and tfoX was induced, the cells became competent for transformation. Several strains of A. actinomycetemcomitans, including different serotypes, as well as rough (adherent) and isogenic smooth (nonadherent) forms were tested. Only our two serotype f strains failed to be transformed. With the other strains, we could easily get transformants with extrachromosomal plasmid DNA when closed circular, replicative plasmid carrying an uptake signal sequence (USS) was used. When a replicative plasmid carrying a USS and cloned DNA from the chromosome of A. actinomycetemcomitans was linearized by digestion with a restriction endonuclease or when genomic DNA was used directly, the outcome was allelic exchange. To facilitate allelic exchange, we constructed a suicide plasmid (pMB78) that does not replicate in A. actinomycetemcomitans and carries a region with two inverted copies of a USS. This vector gave allelic exchange in the presence of cloned and induced tfoX easily and without digestion. Using transposon insertions in cloned katA DNA, we found that as little as 78 bp of homology at one of the ends was sufficient for that end to participate in allelic exchange. The cloning and induction of tfoX makes it possible to transform nearly any strain of A. actinomycetemcomitans, and allelic exchange has proven to be important for site-directed mutagenesis.

Fig 1

Comparison of the tfoX regions of A. actinomycetemcomitans and H. influenzae (Section 2.3). A. Nucleotide sequences of the regions upstream and downstream of the tfoX structural gene, which is delimited by “start” and “stop,” from A. actinomycetemcomitans HK1651 (Roe et al., 2006) and H. influenzae Rd (Zulty and Barcak, 1995). Potential ribosome-binding sites (RBS) and CRP-binding sites (CRP) are indicated. σ⁷⁰-like promoter sequences are marked by “−35” and “−10.” The inverted repeat sequences that can serve as potential intrinsic terminators are shown by convergent arrows. They are followed by six T's shown in italics. The potential CRP and terminator sequences are in bold. Potential RBS, −35, −10, start, and stop sequences are underlined. B. Alignment of the predicted amino acid sequences of the TfoX proteins. Aa is A. actinomycetemcomitans; Hi, H. influenzae. Identical (:) and similar (.) amino acids are indicated. At position 73 the bold 'M' (CU1000) above the bold 'T' (Y4 and HK1651) indicates the amino acid difference in TfoX from CU1000.

Fig 2

Molecular genetic maps (Section 2.3). A. Map of the katA region of A. actinomycetemcomitans Y4N (not to scale) (Thomson et al., 1999) showing uptake signal sequences (USS), positions of transposon IS903ϕkan (Tn) insertions in strains Aa1394 and Aa1395, and the names, positions, and orientations of the primers used for PCR (arrows). B. Relative positions of the tac promoter (tacp), restriction sites, and the USSs in pMB40. The order of restriction sites and the USSs is the same in pMB78. C. Positions and orientations of the EZ::TN<KAN-2> transposon in pMB89, pMB90, pMB91, pMB92 and pMB93. The arrow below each indicates the direction of neo, which arbitrarily orients the transposon.