Novel iron-regulated and Fur-regulated small regulatory RNAs in Aggregatibacter actinomycetemcomitans

Authors

  • J.J. Amarasinghe,

    1.  Department of Microbiology and Immunology, University at Buffalo, State University of New York, Buffalo, NY, USA
    2.  Witebsky Center for Microbial Pathogenesis and Immunology, The School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA
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  • T.D. Connell,

    1.  Department of Microbiology and Immunology, University at Buffalo, State University of New York, Buffalo, NY, USA
    2.  Witebsky Center for Microbial Pathogenesis and Immunology, The School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA
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  • F.A. Scannapieco,

    1.  Department of Oral Biology, School of Dental Medicine, University at Buffalo, State University of New York, Buffalo, NY, USA
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  • E.M. Haase

    1.  Department of Oral Biology, School of Dental Medicine, University at Buffalo, State University of New York, Buffalo, NY, USA
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  • Present address: Division of Infectious Diseases, Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA.

Elaine M. Haase, Department of Oral Biology, University at Buffalo, 109 Foster Hall, 3435 Main Street, Buffalo, NY 14214, USA Tel.: + 1 716 829 2520; fax: + 1 716 829 3942; E-mail: haase@buffalo.edu

Summary

Iron can regulate biofilm formation via non-coding small RNA (sRNA). To determine if iron-regulated sRNAs are involved in biofilm formation by the periodontopathogen Aggregatibacter actinomycetemcomitans, total RNA was isolated from bacteria cultured with iron supplementation or chelation. Transcriptional analysis demonstrated that the expression of four sRNA molecules (JA01–JA04) identified by bioinformatics was significantly upregulated in iron-limited medium compared with iron-rich medium. A DNA fragment encoding each sRNA promoter was able to titrate Escherichia coli ferric uptake regulator (Fur) from a Fur-repressible reporter fusion in an iron uptake regulator titration assay. Cell lysates containing recombinant AaFur shifted the mobility of sRNA-specific DNAs in a gel shift assay. Potential targets of these sRNAs, determined in silico, included genes involved in biofilm formation. The A. actinomycetemcomitans overexpressing JA03 sRNA maintained a rough phenotype on agar, but no longer adhered to uncoated polystyrene or glass, although biofilm determinant gene expression was only modestly decreased. In summary, these sRNAs have the ability to modulate biofilm formation, but their functional target genes remain to be confirmed.

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