PMID:17561357

Citation	Bhattacharjee, MK, Fine, DH and Figurski, DH (2007) tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans. Gene 399:53-64
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.
Links	PubMed PMC2080652 Online version:10.1016/j.gene.2007.04.026
Keywords	Actinobacillus actinomycetemcomitans/genetics; Alleles; Amino Acid Sequence; Cloning, Molecular; Genes, Bacterial/genetics; Genes, Bacterial/physiology; Molecular Sequence Data; Mutagenesis, Site-Directed/methods; Plasmids/genetics; Transformation, Bacterial/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
AGGAD:C9R7I0		GO:0030420: establishment of competence for transformation	IDA: Inferred from Direct Assay:	P	Tables 2 and 3; no transformants were seen in the absence of cloned tfoX. Upon inducing tfoX, the number of transformants increased.	complete CACAO 4936

References

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