@article{milton_draughn_bobay_stowe_olson_feldmann_thompson_myers_santoro_kearns_et al._2020, title={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}, volume={432}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2019.08.019}, abstractNote={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.}, number={2}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Milton, Morgan E. and Draughn, G. Logan and Bobay, Benjamin G. and Stowe, Sean D. and Olson, Andrew L. and Feldmann, Erik A. and Thompson, Richele J. and Myers, Katherine H. and Santoro, Michael T. and Kearns, Daniel B. and et al.}, year={2020}, month={Jan}, pages={343–357} }
 @article{milton_allen_feldmann_bobay_jung_stephens_melander_theisen_zeng_thompson_et al._2017, title={Structure of the Francisella response regulator QseB receiver domain, and characterization of QseB inhibition by antibiofilm 2-aminoimidazole-based compounds}, volume={106}, ISSN={["1365-2958"]}, DOI={10.1111/mmi.13759}, abstractNote={With antibiotic resistance increasing at alarming rates, targets for new antimicrobial therapies must be identified. A particularly promising target is the bacterial two‐component system. Two‐component systems allow bacteria to detect, evaluate and protect themselves against changes in the environment, such as exposure to antibiotics and also to trigger production of virulence factors. Drugs that target the response regulator portion of two‐component systems represent a potent new approach so far unexploited. Here, we focus efforts on the highly virulent bacterium Francisella tularensis tularensis. Francisella contains only three response regulators, making it an ideal system to study. In this study, we initially present the structure of the N‐terminal domain of QseB, the response regulator responsible for biofilm formation. Subsequently, using binding assays, computational docking and cellular studies, we show that QseB interacts with2‐aminoimidazole based compounds that impede its function. This information will assist in tailoring compounds to act as adjuvants that will enhance the effect of antibiotics.}, number={2}, journal={MOLECULAR MICROBIOLOGY}, author={Milton, Morgan E. and Allen, C. Leigh and Feldmann, Erik A. and Bobay, Benjamin G. and Jung, David K. and Stephens, Matthew D. and Melander, Roberta J. and Theisen, Kelly E. and Zeng, Daina and Thompson, Richele J. and et al.}, year={2017}, month={Oct}, pages={223–235} }
 @article{feldmann_cavanagh_2015, title={Teaching old drugs new tricks: Addressing resistance in Francisella}, volume={6}, ISSN={["2150-5608"]}, DOI={10.1080/21505594.2015.1053689}, abstractNote={Francisella tularensis is a Gram-negative bacterial pathogen and causative agent of the disease tularemia, also known as “rabbit fever.” The Centers for Disease Control and Prevention (CDC) lists the virulent form of F. tularensis as a Tier 1 pathogen based on its ease in aerosolizing and the lethal damage an inhaled infection can cause in humans. Because of its high pathogenicity and aerosolization ease, F. tularensis is classified as a Category A select agent and represents a significant potential threat as a biological weapon. Especially concerning is the emergence of multi-drug resistant (MDR) Francisella strains adding to the already dire public health and safety concerns. Discovery of therapeutic strategies that avoid conferring resistance is crucial for any long-term solution to address the problem of MDR bacteria and enhances the motivation for Francisella research. Francisella novicida is a useful model strain for studying virulence, biofilm formation, and drug resistance in the far more virulent strain, F. tularensis. Unlike other bacteria, F. novicida relies on only two intact 2-component systems (TCSs) and two orphaned members for its virulence and resistive strategies, making any of these select members particularly attractive drug targets. Francisella, like all bacteria, utilize TCS signaling pathways to respond to environmental stimuli through concerted communication between a histidine sensor kinase and a response regulator target. If the TCS signaling components malfunction, bacterial defense mechanisms are compromised, rendering them susceptible to environmental stress. When TCS functionality is specifically targeted, using small molecule drugs or biologics for example, even MDR bacteria can once again show vulnerability to the same antibiotics to which they had previously acquired resistance. Similar to F. novicida, F. tularensis encodes orphaned TCS components but the identity and number of these differ depending on the substrain. For example, the highly virulent strain F. tularensis Schu S4 encodes QseC, PmrA/QseB, KdpD, and FTT1543 (the conserved homolog of FTN1452 in F. novicida) all as orphans (QseC and KdpD are sensor kinases; PmrA/QseB and FTT1543 are response regulators). TCS signal transduction pathways generally make attractive targets for the development of anti-infective therapeutics since so many cellular processes rely on the downstream activities from both sensor kinases and response regulators. The accelerated work over the last 20 years is indicative of their potential for use as therapeutics and has been reviewed elsewhere. Recently, efforts have been made to target response regulator proteins in a variety of systems to thwart bacterial resistance and the onset of virulence. For example, PmrA/QseB is the lone response regulator orphan in F. novicida and is an important regulator of biofilm formation across the Francisella genus. PmrA/QseB has been shown to be required for virulence and proper expression of Francisella pathogenicity island (FPI) virulence factors, many of which themselves are also required for virulence and infectivity. PmrA/QseB upregulates the transcription of FPI genes, most notably the levels of intracellular growth locus C (iglC). The activation of PmrA/QseB is mediated by phosphorylation from an upstream sensor kinase, the identity of which is still unclear, but this action could be accomplished by KdpD or even multiple kinases. A proposed model suggests that Francisella sensor kinases may not necessarily discriminate for a single response regulator partner, but rather phosphorylate their targets more “promiscuously”. Regardless, PmrA/ QseB appears to form a complex with another virulence factor, MglA, a regulator of IglC. MglA has subsequently been suggested to form a complex with yet another virulence factor, SspA. Furthermore, transcription of FPI appears to be controlled by the phosphorylation/activation of PmrA/QseB which then recruits MglA-SspA together with RNA-polymerase to bind target gene promoters and upregulate the virulence pathway. However, it has historically been the sensor kinases, not the response regulators, that have attracted the most attention for therapeutic intervention. QseC from Francisella is a sensor kinase found in many bacteria including other biothreat pathogens like MDR Salmonella, Coxiella brunettii, and enterohemorrhagic E. coli (EHEC). The operon for QseC in F. novicida does not encode a paired response regulator, unlike KdpD (paired response regulator is KdpE) and FTN1453 (paired response regulator is FTN1452), and is thus considered orphaned. However despite this, QseC has been shown together with PmrA/QseB to regulate biofilm development in F. novicida and is required for virulence. QseC in Francisella is homologous to QseC in E. coli,}, number={5}, journal={VIRULENCE}, author={Feldmann, Erik A. and Cavanagh, John}, year={2015}, month={Jul}, pages={414–416} }