Mlc is a transcriptional activator with a key role in integrating cyclic amp receptor protein and integration host factor regulation of leukotoxin RNA synthesis in aggregatibacter actinomycetemcomitans

Catherine Childress, Leigh A. Feuerbacher, Linda Phillips, Alex Burgum, David J Kolodrubetz

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cyclic AMP (cAMP) receptor protein (CRP) indirectly increases ltxA expression, but the intermediary regulator is unknown. Integration host factor (IHF) binds to and represses the leukotoxin promoter, but neither CRP nor IHF is responsible forthe anaerobic induction of ltxA RNA synthesis. Thus, we have undertaken studies to identify other regulators of leukotoxin transcription and to demonstrate how these proteins work together to modulate leukotoxin synthesis. First, analyses of ltxA RNA expression from defined leukotoxin promoter mutations in the chromosome identify positions-69 to -35 as the key control region and indicate that an activator protein modulates leukotoxin transcription. We show that Mlc, which isa repressor in Escherichia coli, functions as a direct transcriptional activator in A. actinomycetemcomitans; an mlc deletion mutant reduces leukotoxin RNA synthesis, and recombinant Mlc protein binds specifically at the -68 to -40 region of the leukotoxin promoter. Furthermore, we show that CRP activates ltxA expression indirectly by increasing the levels of Mlc. Analyses of δmlc, δihf, and δihf δmlc strains demonstrate that Mlc can increase RNA polymerase (RNAP) activity directly and that IHF represses ltxA RNA synthesis mainly by blocking Mlc binding. Finally, a δihf δmlc mutant stillinduces ltxA during anaerobic growth, indicating that there are additional factors involved in leukotoxin transcriptional regulation. A model for the coordinated regulation of leukotoxin transcription is presented.

Original languageEnglish (US)
Pages (from-to)2284-2297
Number of pages14
JournalJournal of Bacteriology
Volume195
Issue number10
DOIs
StatePublished - May 2013

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Integration Host Factors
Aggregatibacter actinomycetemcomitans
RNA
Cyclic AMP Receptor Protein
Proteins
leukotoxin
DNA-Directed RNA Polymerases
Operon
Growth
Recombinant Proteins
Genetic Promoter Regions
Chromosomes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Mlc is a transcriptional activator with a key role in integrating cyclic amp receptor protein and integration host factor regulation of leukotoxin RNA synthesis in aggregatibacter actinomycetemcomitans. / Childress, Catherine; Feuerbacher, Leigh A.; Phillips, Linda; Burgum, Alex; Kolodrubetz, David J.

In: Journal of Bacteriology, Vol. 195, No. 10, 05.2013, p. 2284-2297.

Research output: Contribution to journalArticle

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abstract = "Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cyclic AMP (cAMP) receptor protein (CRP) indirectly increases ltxA expression, but the intermediary regulator is unknown. Integration host factor (IHF) binds to and represses the leukotoxin promoter, but neither CRP nor IHF is responsible forthe anaerobic induction of ltxA RNA synthesis. Thus, we have undertaken studies to identify other regulators of leukotoxin transcription and to demonstrate how these proteins work together to modulate leukotoxin synthesis. First, analyses of ltxA RNA expression from defined leukotoxin promoter mutations in the chromosome identify positions-69 to -35 as the key control region and indicate that an activator protein modulates leukotoxin transcription. We show that Mlc, which isa repressor in Escherichia coli, functions as a direct transcriptional activator in A. actinomycetemcomitans; an mlc deletion mutant reduces leukotoxin RNA synthesis, and recombinant Mlc protein binds specifically at the -68 to -40 region of the leukotoxin promoter. Furthermore, we show that CRP activates ltxA expression indirectly by increasing the levels of Mlc. Analyses of δmlc, δihf, and δihf δmlc strains demonstrate that Mlc can increase RNA polymerase (RNAP) activity directly and that IHF represses ltxA RNA synthesis mainly by blocking Mlc binding. Finally, a δihf δmlc mutant stillinduces ltxA during anaerobic growth, indicating that there are additional factors involved in leukotoxin transcriptional regulation. A model for the coordinated regulation of leukotoxin transcription is presented.",
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AB - Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cyclic AMP (cAMP) receptor protein (CRP) indirectly increases ltxA expression, but the intermediary regulator is unknown. Integration host factor (IHF) binds to and represses the leukotoxin promoter, but neither CRP nor IHF is responsible forthe anaerobic induction of ltxA RNA synthesis. Thus, we have undertaken studies to identify other regulators of leukotoxin transcription and to demonstrate how these proteins work together to modulate leukotoxin synthesis. First, analyses of ltxA RNA expression from defined leukotoxin promoter mutations in the chromosome identify positions-69 to -35 as the key control region and indicate that an activator protein modulates leukotoxin transcription. We show that Mlc, which isa repressor in Escherichia coli, functions as a direct transcriptional activator in A. actinomycetemcomitans; an mlc deletion mutant reduces leukotoxin RNA synthesis, and recombinant Mlc protein binds specifically at the -68 to -40 region of the leukotoxin promoter. Furthermore, we show that CRP activates ltxA expression indirectly by increasing the levels of Mlc. Analyses of δmlc, δihf, and δihf δmlc strains demonstrate that Mlc can increase RNA polymerase (RNAP) activity directly and that IHF represses ltxA RNA synthesis mainly by blocking Mlc binding. Finally, a δihf δmlc mutant stillinduces ltxA during anaerobic growth, indicating that there are additional factors involved in leukotoxin transcriptional regulation. A model for the coordinated regulation of leukotoxin transcription is presented.

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