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Thermodynamic and molecular analysis of the AbrB-binding sites within the phyC-region of Bacillus amyloliquefaciens FZB45

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Abstract

AbrB is a global regulator of transition state that is known to repress more than 100 genes in Bacillus species. Although AbrB is involved in the regulation of most cellular processes, a conserved binding motif seems to be elusive. Thus, the mechanism of AbrB-mediated transcriptional control is still unclear. In our previous work we identified two separate AbrB-binding sites within phytase gene region (phyC) of Bacillus amyloliquefaciens FZB45, whose integrity is essential for repression. Comparable architecture of AbrB-binding sites is also described for tycA that encodes an antibiotic synthesis enzyme. Considering the size of the AbrB tetramer (56 kDa) and other AbrB binding motifs (~20 to 98 bp) we hypothesized preferred binding positions within both AbrB sites of phyC that exhibit higher affinities to AbrB. Thus, we used surface plasmon resonance (SPR) to study the binding kinetics between AbrB and 40-bp ds-oligonucleotides that were derived from both binding sites. Surface plasmon resonance sensorgrams revealed strong binding kinetics that showed nearly no dissociation and positive cooperativity of the AbrB-DNA interaction to the whole AbrB-binding site 2 and to a small part of AbrB-binding site 1. Using chemically modified DNA we found bases contacting AbrB mainly at one face of the DNA-helix within a core region separated by one helical turn each. High content of modified guanines presented in the control reaction of the KMnO4 interference assay indicated distortion of the DNA-structure of phyC. In vitro transcription assays and base substitutions within the core region support this idea and the cooperativity of AbrB binding.

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Acknowledgments

Financial support given within the Deutsche Forschungsgemeinschaft, DFG (Grant BO 1113/9-1 to R.B.), is gratefully acknowledged. The authors thank Tobias Werther from FMP (Leibniz-Institut für Molekulare Pharmakologie) for introduction to the Biacore-technique. The authors also thank Christiane Müller for technical support and sequencing and Alexandra Koumoutsi for critical reading of the manuscript.

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Authors and Affiliations

  1. Institut für Biologie, Humboldt Universität zu Berlin, AG Bakteriengenetik, Chausseestrasse 117, 10115, Berlin, Germany

    Svetlana Neubauer, Rainer Borriss & Oliwia Makarewicz

  2. Universitätsklinikum Jena, Klinik für Innere Medizin II, Sektion Klinische Infektiologie, Erlanger Allee 101, 07747, Jena, Germany

    Oliwia Makarewicz

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  1. Svetlana Neubauer
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  2. Rainer Borriss
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  3. Oliwia Makarewicz
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Correspondence to Oliwia Makarewicz.

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Communicated by M. Hecker.

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Neubauer, S., Borriss, R. & Makarewicz, O. Thermodynamic and molecular analysis of the AbrB-binding sites within the phyC-region of Bacillus amyloliquefaciens FZB45. Mol Genet Genomics 287, 111–122 (2012). https://doi.org/10.1007/s00438-011-0666-4

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