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Volume 154, Issue 2

Induction of leukotoxin expression by IS and Free

Abstract

Most strains express relatively low levels of leukotoxin, encoded by the operon. However, several strains isolated from patients with localized aggressive periodontitis are hyperleukotoxic and transcribe the operon at high levels. These strains possess a copy of IS in the promoter and previous studies have suggested that the presence of the insertion sequence increases transcription by uncoupling a -acting negative regulator of expression from the basal elements of the promoter. However, we now report that replacing IS with an equal length of random sequence has little effect on transcriptional activity of the promoter, suggesting that the physical displacement of the negative regulatory element does not contribute to the hyperleukotoxic phenotype of IS-containing strains. Instead, we show that a −10-like element upstream of the transposase ORF of IS is required for increased transcriptional activity of the promoter. Site-specific mutation of the −10 sequence, or reversing the orientation of IS relative to the basal promoter elements, reduced transcriptional activity to levels exhibited by the native promoter. However, no increase in transcription was observed when IS was recombinantly inserted into a promoter that contained a truncated copy of , suggesting that an intact may also be required for IS-mediated induction of . Therefore, to determine if functions as a regulator of expression, three independent -promoter–-reporter constructs containing frameshift mutations in were analysed. Each exhibited significantly lower expression of -galactosidase than the control reporter with intact . In addition, OrfA protein was shown, by mobility shift electrophoresis, to interact with the promoter at or downstream of the −35 sequence. These results suggest that a potential transposase promoter and the OrfA polypeptide may modulate leukotoxin expression in hyperleukotoxic strains containing IS.

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Keyword(s): EMSA, electrophoretic mobility shift assay and RTX, repeats in toxin
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2008-02-01
2024-04-18
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Induction of Aggregatibacter actinomycetemcomitans leukotoxin expression by IS1301 and orfA
Microbiology 154, 528 (2008); https://doi.org/10.1099/mic.0.2007/012195-0
/content/journal/micro/10.1099/mic.0.2007/012195-0
/content/journal/micro/10.1099/mic.0.2007/012195-0
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