CIS-ELEMENTS AND TRANS FACTORS ARE BOTH IMPORTANT IN STRAIN-SPECIFIC REGULATION OF THE LEUKOTOXIN GENE IN ACTINOBACILLUS-ACTINOMYCETEMCOMITANS

Actinobacillus actinomycetemcomitans, the etiologic agent of localized juvenile periodontitis, produces a potent leukotoxin that kills human neutrophils. The production of leukotoxin RNA can vary more than 50-f old among isolates of A. actinomycetemcomitans, and strains expressing high levels of leukotoxin RNA are most often found at sites of period ontal disease, To assess the relative contributions of transcription f actors and promoter sequences in setting the disparate levels of leuko toxin RNA found, we have undertaken classical cis/trans analyses, Firs t, the leukotoxin promoter regions from moderately leukotoxic (Y4) and minimally leukotoxic (ATCC 33384) strains of A, actinomycetemcomitans were cloned, sequenced, and compared with the previously sequenced le ukotoxin promoter region of the high-producer strain JP2. The Y4 and A TCC 33384 promoter regions each contain a 528-bp segment that is absen t from JP2, Interestingly, the analysis of various deletion constructs in A. actinomycetemcomitans indicated that Y4, despite the large inse rtion, initiates leukotoxin RNA synthesis at the same promoter as JP2 does, To perform cis/trans analyses, these three leukotoxin promoter r egions were cloned into a plasmid upstream of the reporter gene beta-g alactosidase, Each plasmid was transformed into JP2, Y4, and ATCC 3338 4, and the beta-galactosidase levels were determined, The results indi cated that the sequences responsible for down-regulating leukotoxin RN A levels in Y4 relative to JP2 are found within the transcribed region of the Y4 leukotoxin operon, Importantly, in ATCC 33384, strain-speci fic trans factors and promoter sequence differences are equally signif icant in determining the lower levels of leukotoxin RNA, We hypothesiz e that either strain ATCC 33384 has a negative regulatory protein (whi ch is missing or mutated in JP2/Y4) or that JP2 and Y4 carry an activa tor that is missing or mutated in ATCC 33384.