Transformation of <i>Aggregatibacter actinomycetemcomitans</i> with Green Fluorescent Protein

Abstract

Green fluorescent protein (GFP) is one of the most useful biological markers for monitoring gene expression and the localization of particular cells. We constructed a GFPexpressing Aggregatibacter actinomycetemcomitans transformant by transposon insertion.This anaerobic, oral, gram-negative bacterium is thought to be the major causative agent in localized aggressive periodontitis and possesses the gene coding for leukotoxin (ltx). As this gene is stably expressed, the region extending from the ltx promoter to the initiation codon was amplified using PCR and temporarily cloned into a small Escherichia coli plasmid, pResKm. The DNA fragment containing the GFP gene(gfp)ORF was excised from the commercially available plasmid pAcGFP1-C1, purified by gel electrophoresis, and inserted downstream of the ltx initiation codon in the correct reading frame. This gene fusion was further cloned into the NotI site of the commercially available shuttle plasmid, pUTminiTn5, which contains the gene coding for the transposase, and then introduced into the chromosome of A. actinomycetemcomitans strain HK1651 by electroporation.Transformants were isolated by screening for the appropriate antibiotic resistance. FACS analysis revealed that the final A. actinomycetemcomitans-GFP integrant exhibited slightly more intense fluorescence than wild-type A. actinomycetemcomitans. These data demonstrate that transposon-mediated chromosomal integration is a useful approach for constructing mutant A. actinomycetemcomitans strains.