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. 1996 Sep;64(9):3451–3460. doi: 10.1128/iai.64.9.3451-3460.1996

cis Elements and trans factors are both important in strain-specific regulation of the leukotoxin gene in Actinobacillus actinomycetemcomitans.

D Kolodrubetz 1, J Spitznagel Jr 1, B Wang 1, L H Phillips 1, C Jacobs 1, E Kraig 1
PMCID: PMC174248  PMID: 8751884

Abstract

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-fold among isolates of A. actinomycetemcomitans, and strains expressing high levels of leukotoxin RNA are most often found at sites of periodontal disease. To assess the relative contributions of transcription factors and promoter sequences in setting the disparate levels of leukotoxin RNA found, we have undertaken classical cis/trans analyses. First, 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 sequences leukotoxin promoter region of the high-producer strain JP2. The Y4 and ATCC 33384 promoter regions each contain a 528-bp segment that is absent from JP2. Interestingly, the analysis of various deletion constructs in A. actinomycetemcomitans indicated that Y4, despite the large insertion, initiates leukotoxin RNA synthesis at the same promoter as JP2 does. To perform cis/trans analyses, these three leukotoxin promoter regions were cloned into a plasmid upstream of the reporter gene beta-galactosidase. Each plasmid was transformed into JP2, Y4, and ATCC 33384, and the beta-galactosidase levels were determined. The results indicated that the sequences responsible for down-regulating leukotoxin RNA levels in Y4 relative to JP2 are found within the transcribed region of the Y4 leukotoxin operon. Importantly, in ATCC 33384, strain-specific trans factors and promoter sequence differences are equally significant in determining the lower levels of leukotoxin RNA. We hypothesize that either strain ATCC 33384 has a negative regulatory protein (which is missing or mutated in JP2/Y4) or that JP2 and Y4 carry an activator that is missing or mutated in ATCC 33384.

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Selected References

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