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

Abstract

AbstracttfoX (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.

Defining Genetic Fitness Determinants and Creating Genomic Resources for an Oral Pathogen

“…4). The remaining 2 genes, however, encoded less well recognized biofilm determinants-leukotoxin (ltxB [19]) and the DNA uptake transcriptional regulator TfoX (71). Leukotoxin is widely regarded as the primary virulence factor of A. actinomycetemcomitans, as it lyses both white and red blood cells (20), but recently it was also shown to control adherence.…”

CRP Binding and Transcription Activation at CRP-S Sites

“…9 All H. influenzae promoters with CRP-S sites also require the protein Sxy for transcription activation, raising the possibility that Sxy assists CRP binding to CRP-S sites and/or RNAP recruitment. 13 Although the abovementioned three bacterial families all have Sxy homologs, and extensive genetic studies have confirmed Sxy's role as a regulator of competence genes, 5,[13][14][15][16][17][18][19] direct characterization of its action at CRP-S promoters has been stymied by the toxicity and intractability of this small protein. To better understand CRP binding to CRP-S sites, we have conducted a detailed analysis of H. influenzae (Hi) CRP and E. coli (Ec)CRP binding and transcriptional activation at native and synthetic CRP sites.…”

A Comparison of Aggregatibacter actinomycetemcomitans (Aa) Virulence Traits in a Rat Model for Periodontal Disease

“…This plasmid was named HS66. Aa strain IDH781Nal (pMB7) [29] was transformed with HS66 DNA using an inducible tfox system [29]. The position of the transposon in the bacterial strain was verified by sequencing, analysis using primers CC4 and CB6.…”