tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans.
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.
Bhattacharjee
Mrinal K
MK
Department of Microbiology, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032, USA.
Fine
Daniel H
DH
Figurski
David H
DH
D016976:Aggregatibacter actinomycetemcomitans / Q000235:genetics*; D000483:Alleles; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D014169:Transformation, Bacterial / Q000235:genetics*