Browsing by Author "Olson, Andrew L"

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    A DNA mimic: the structure and mechanism of action for the anti-repressor protein AbbA.
    (Journal of molecular biology, 2014-05) Tucker, Ashley T; Bobay, Benjamin G; Banse, Allison V; Olson, Andrew L; Soderblom, Erik J; Moseley, M Arthur; Thompson, Richele J; Varney, Kristen M; Losick, Richard; Cavanagh, John
    Bacteria respond to adverse environmental conditions by switching on the expression of large numbers of genes that enable them to adapt to unfavorable circumstances. In Bacillus subtilis, many adaptive genes are under the negative control of the global transition state regulator, the repressor protein AbrB. Stressful conditions lead to the de-repression of genes under AbrB control. Contributing to this de-repression is AbbA, an anti-repressor that binds to and blocks AbrB from binding to DNA. Here, we have determined the NMR structure of the functional AbbA dimer, confirmed that it binds to the N-terminal DNA-binding domain of AbrB, and have provided an initial description for the interaction using computational docking procedures. Interestingly, we show that AbbA has structural and surface characteristics that closely mimic the DNA phosphate backbone, enabling it to readily carry out its physiological function.
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    ¹H, ¹³C, and ¹⁵N resonance assignments and secondary structure prediction of the full-length transition state regulator AbrB from Bacillus anthracis.
    (Biomolecular NMR assignments, 2012-04) Olson, Andrew L; Bobay, Benjamin G; Melander, Christian; Cavanagh, John
    The AbrB protein is a transcription factor that regulates the expression of numerous essential genes during the cells transition phase state. AbrB from Bacillus anthracis is, nototriously, the principal protein responsible for anthrax toxin gene expression and is highly homologous to the much-studied AbrB protein from Bacillus subtilis having 85% sequence identity and the ability to regulate the same target promoters. Here we report backbone and sidechain resonance assignments and secondary structure prediction for the full-length AbrB protein from B. anthracis.
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    Identification of BfmR, a response regulator involved in biofilm development, as a target for a 2-Aminoimidazole-based antibiofilm agent.
    (Biochemistry, 2012-12) Thompson, Richele J; Bobay, Benjamin G; Stowe, Sean D; Olson, Andrew L; Peng, Lingling; Su, Zhaoming; Actis, Luis A; Melander, Christian; Cavanagh, John
    2-Aminoimidazoles (2AIs) have been documented to disrupt bacterial protection mechanisms, including biofilm formation and genetically encoded antibiotic resistance traits. Using Acinetobacter baumannii, we provide initial insight into the mechanism of action of a 2AI-based antibiofilm agent. Confocal microscopy confirmed that the 2AI is cell permeable, while pull-down assays identified BfmR, a response regulator that is the master controller of biofilm formation, as a target for this compound. Binding assays demonstrated specificity of the 2AI for response regulators, while computational docking provided models for 2AI-BfmR interactions. The 2AI compound studied here represents a unique small molecule scaffold that targets bacterial response regulators.
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    Structure and DNA-binding traits of the transition state regulator AbrB.
    (Structure (London, England : 1993), 2014-11) Olson, Andrew L; Tucker, Ashley T; Bobay, Benjamin G; Soderblom, Erik J; Moseley, M Arthur; Thompson, Richele J; Cavanagh, John
    The AbrB protein from Bacillus subtilis is a DNA-binding global regulator controlling the onset of a vast array of protective functions under stressful conditions. Such functions include biofilm formation, antibiotic production, competence development, extracellular enzyme production, motility, and sporulation. AbrB orthologs are known in a variety of prokaryotic organisms, most notably in all infectious strains of Clostridia, Listeria, and Bacilli. Despite its central role in bacterial response and defense, its structure has been elusive because of its highly dynamic character. Orienting its N- and C-terminal domains with respect to one another has been especially problematic. Here, we have generated a structure of full-length, tetrameric AbrB using nuclear magnetic resonance, chemical crosslinking, and mass spectrometry. We note that AbrB possesses a strip of positive electrostatic potential encompassing its DNA-binding region and that its C-terminal domain aids in DNA binding.
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    The Solution Structures and Interaction of SinR and SinI: Elucidating the Mechanism of Action of the Master Regulator Switch for Biofilm Formation in Bacillus subtilis.
    (Journal of molecular biology, 2020-01) Milton, Morgan E; Draughn, G Logan; Bobay, Benjamin G; Stowe, Sean D; Olson, Andrew L; Feldmann, Erik A; Thompson, Richele J; Myers, Katherine H; Santoro, Michael T; Kearns, Daniel B; Cavanagh, John
    Bacteria have developed numerous protection strategies to ensure survival in harsh environments, with perhaps the most robust method being the formation of a protective biofilm. In biofilms, bacterial cells are embedded within a matrix that is composed of a complex mixture of polysaccharides, proteins, and DNA. The gram-positive bacterium Bacillus subtilis has become a model organism for studying regulatory networks directing biofilm formation. The phenotypic transition from a planktonic to biofilm state is regulated by the activity of the transcriptional repressor, SinR, and its inactivation by its primary antagonist, SinI. In this work, we present the first full-length structural model of tetrameric SinR using a hybrid approach combining high-resolution solution nuclear magnetic resonance (NMR), chemical cross-linking, mass spectrometry, and molecular docking. We also present the solution NMR structure of the antagonist SinI dimer and probe the mechanism behind the SinR-SinI interaction using a combination of biochemical and biophysical techniques. As a result of these findings, we propose that SinI utilizes a residue replacement mechanism to block SinR multimerization, resulting in diminished DNA binding and concomitant decreased repressor activity. Finally, we provide an evidence-based mechanism that confirms how disruption of the SinR tetramer by SinI regulates gene expression.
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    The Structure of the Biofilm-controlling Response Regulator BfmR from Acinetobacter baumannii Reveals Details of Its DNA-binding Mechanism.
    (Journal of molecular biology, 2018-03) Draughn, G Logan; Milton, Morgan E; Feldmann, Erik A; Bobay, Benjamin G; Roth, Braden M; Olson, Andrew L; Thompson, Richele J; Actis, Luis A; Davies, Christopher; Cavanagh, John
    The rise of drug-resistant bacterial infections coupled with decreasing antibiotic efficacy poses a significant challenge to global health care. Acinetobacter baumannii is an insidious, emerging bacterial pathogen responsible for severe nosocomial infections aided by its ability to form biofilms. The response regulator BfmR, from the BfmR/S two-component system, is the master regulator of biofilm initiation in A. baumannii and is a tractable therapeutic target. Here we present the structure of A. baumannii BfmR using a hybrid approach combining X-ray crystallography, nuclear magnetic resonance spectroscopy, chemical crosslinking mass spectrometry, and molecular modeling. We also show that BfmR binds the previously proposed bfmRS promoter sequence with moderate affinity. While BfmR shares many traits with other OmpR/PhoB family response regulators, some unusual properties were observed. Most importantly, we observe that when phosphorylated, BfmR binds this promoter sequence with a lower affinity than when not phosphorylated. All other OmpR/PhoB family members studied to date show an increase in DNA-binding affinity upon phosphorylation. Understanding the structural and biochemical mechanisms of BfmR will aid in the development of new antimicrobial therapies.