Associate Professor Lynette Cegelski's research is inspired by the challenge and importance of elucidating chemical structure and function in biological systems and the need for new and unconventional strategies to treat infectious diseases. Cegelski completed her undergraduate studies in Chemistry at SUNY-Binghamton, New York (B.S. summa cum laude and Phi Beta Kappa 1998), where she participated in research to determine the microtubule-bound conformation of the anti-cancer drug Taxol by REDOR solid-state NMR. This formative experience motivated her move to Washington University to conduct her PhD training in the laboratory of Professor Jacob Schaefer (Ph.D. Chemistry 2004). She investigated bacterial and plant macromolecular and whole-cell systems using solid-state NMR, including examining the mode of action of the antibiotic oritavancin and investigating photosynthesis and photorespiration in soybean leaves with 13CO2 and 15N labeling. She introduced the use of 4-frequency TEDOR-REDOR measurements in whole cells for the first time. She trained in Microbiology and Infectious Disease research as a postdoctoral fellow in Molecular Microbiology at the Washington University School of Medicine, working with Professor Scott Hultgren. There, she defined amyloid contributions to E. coli biofilms and introduced the first small-molecule inhibitors of functional amyloid assembly in bacteria. She joined the faculty of the Stanford Chemistry Department in 2008. The Cegelski Research program integrates chemistry, biology, and physics to investigate the assembly and function of macromolecular and whole-cell systems. They are revealing new bacterial structures, uncovering fundamental parameters of chemical composition and architecture in complex biofilm assemblies, and identifying new anti-infectives and anti-infective strategies. Cegelski's work has garnered early career awards, including the Burroughs Wellcome Career Award at the Scientific Interface, the 2010 NIH Director’s New Innovator Award, the National Science Foundation CAREER Award, and the Presidential Early Career Award for Scientists and Engineers (PECASE).

Academic Appointments

Honors & Awards

  • Presidential Early Career Award for Scientists and Engineers, National Science Foundation (2019)
  • ICMRBS Founder’s Medal, International Council on Magnetic Resonance in Biological Systems (2018)
  • NSF CAREER Award, National Science Foundation (2015)
  • Hellman Faculty Scholar Award, Hellman Fellows Fund (2012)
  • Terman Fellowship, Stanford University (2011)
  • NIH Director's New Innovator Award, National Institutes of Health (2010)
  • Career Award at the Scientific Interface, Burroughs Wellcome Fund (2008)
  • Terman Fellowship, Stanford University (2008)

Professional Education

  • Postdoc, Washington University School of Medicine, Molecular Microbiology (2008)
  • PhD, Washington University, Chemistry (2004)
  • BS, Binghamton University, SUNY, Chemistry (1998)


  • Huttner, M.; Wender, P; Cegelski, L.; Zang, Xiaoyu; Antonoplis, A. "United States Patent 62/633,368 (WO2019165051A1) Composition and method for new antimicrobial agents with secondary mode of action", Leland Stanford Junior University, Aug 29, 2019
  • Lynette Cegelski, Wiriya Thongsomboon. "United States Patent WO2018035411A1 Production and Use of Phosphoethanolamine Cellulose and Derivatives", Leland Stanford Junior University, Feb 22, 2018
  • Lynette Cegelski, Ji Youn Lim. "United States Patent 9,271,493 Methods for Microbial Biofilm Destruction", Leland Stanford Junior University, Mar 1, 2016

Current Research and Scholarly Interests

Our research program is inspired by the challenge and importance of elucidating chemical structure and function in complex biological systems and the need for new strategies to treat infectious diseases. The genomics and proteomics revolutions have been enormously successful in generating crucial "parts lists" for biological systems. Yet, for many fascinating systems, formidable challenges exist in building complete descriptions of how the parts function and assemble into macromolecular complexes and whole-cell factories. We have introduced uniquely enabling problem-solving approaches integrating solid-state NMR spectroscopy with microscopy and biochemical and biophysical tools to determine atomic- and molecular-level detail in complex macromolecular assemblies and whole cells and biofilms. We are uncovering new chemistry and new chemical structures produced in nature. We identify small molecules that influence bacterial assembly processes and use these in chemical genetics approaches to learn about bacterial cell wall, amyloid and biofilm assembly.

Translationally, we have launched a collaborative antibacterial drug design program integrating synthesis, chemical biology, and mechanistic biochemistry and biophysics directed at the discovery and development of new antibacterial therapeutics targeting difficult-to-treat bacteria.

2023-24 Courses

Stanford Advisees

Graduate and Fellowship Programs

All Publications

  • CPMAS NMR platform for direct compositional analysis of mycobacterial cell-wall complexes and whole cells. Journal of magnetic resonance open Liu, X., Brčić, J., Cassell, G. H., Cegelski, L. 2023; 16-17


    Tuberculosis and non-tuberculosis mycobacterial infections are rising each year and often result in chronic incurable disease. Important antibiotics target cell-wall biosynthesis, yet some mycobacteria are alarmingly resistant or tolerant to currently available antibiotics. This resistance is often attributed to assumed differences in composition of the complex cell wall of different mycobacterial strains and species. However, due to the highly crosslinked and insoluble nature of mycobacterial cell walls, direct comparative determinations of cell-wall composition pose a challenge to analysis through conventional biochemical analyses. We introduce an approach to directly observe the chemical composition of mycobacterial cell walls using solid-state NMR spectroscopy. 13C CPMAS spectra are provided of individual components (peptidoglycan, arabinogalactan, and mycolic acids) and of in situ cell-wall complexes. We assigned the spectroscopic contributions of each component in the cell-wall spectrum. We uncovered a higher arabinogalactan-to-peptidoglycan ratio in the cell wall of M. abscessus, an organism noted for its antibiotic resistance, relative to M. smegmatis. Furthermore, differentiating influences of different types of cell-wall targeting antibiotics were observed in spectra of antibiotic-treated whole cells. This platform will be of value in evaluating cell-wall composition and antibiotic activity among different mycobacteria and in considering the most effective combination treatment regimens.

    View details for DOI 10.1016/j.jmro.2023.100127

    View details for PubMedID 38125335

    View details for PubMedCentralID PMC10732466

  • Conjugation of Vancomycin with a Single Arginine Improves Efficacy against Mycobacteria by More Effective Peptidoglycan Targeting. Journal of medicinal chemistry Brčić, J., Tong, A., Wender, P. A., Cegelski, L. 2023


    Drug resistant bacterial infections have emerged as one of the greatest threats to public health. The discovery and development of new antimicrobials and anti-infective strategies are urgently needed to address this challenge. Vancomycin is one of the most important antibiotics for the treatment of Gram-positive infections. Here, we introduce the vancomycin-arginine conjugate (V-R) as a highly effective antimicrobial against actively growing mycobacteria and difficult-to-treat mycobacterial biofilm populations. Further improvement in efficacy through combination treatment of V-R to inhibit peptidoglycan synthesis and ethambutol to inhibit arabinogalactan synthesis underscores the ability to identify compound synergies to more effectively target the Achilles heel of the cell-wall assembly. Moreover, we introduce mechanistic activity data and a molecular model derived from a d-Ala-d-Ala-bound vancomycin structure that we hypothesize underlies the molecular basis for the antibacterial improvement attributed to the arginine modification that is specific to peptidoglycan chemistry employed by mycobacteria and distinct from Gram-positive pathogens.

    View details for DOI 10.1021/acs.jmedchem.3c00565

    View details for PubMedID 37477249

  • In vivo targeting of E. coli with vancomycin-arginine. Antimicrobial agents and chemotherapy Neville, L. F., Shalit, I. n., Warn, P. A., Scheetz, M. H., Sun, J. n., Chosy, M. B., Wender, P. A., Cegelski, L. n., Rendell, J. T. 2021


    The ability of vancomycin-arginine (V-r) to extend the spectrum of activity of glycopeptides to gram-negative bacteria was investigated. Its MIC towards E. coli including β-lactamase expressing Ambler classes A, B, and D was 8-16μg/ml. Addition of 8×MIC V-r to E. coli was acutely bactericidal and associated with a low frequency of resistance (< 2.3×10-10). In vivo, V-r markedly reduced E. coli burden by >7 log10 CFU/g in a thigh muscle model. These data warrant further development of V-r in combatting E. coli, including resistant forms.

    View details for DOI 10.1128/AAC.02416-20

    View details for PubMedID 33468474

  • Vancomycin-Arginine Conjugate Inhibits Growth of Carbapenem-Resistant E. coli and Targets Cell-Wall Synthesis. ACS chemical biology Antonoplis, A. n., Zang, X. n., Wegner, T. n., Wender, P. A., Cegelski, L. n. 2019


    The emergence of multi-drug-resistant Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae, is a major health problem that necessitates the development of new antibiotics. Vancomycin inhibits cell-wall synthesis in Gram-positive bacteria but is generally ineffective against Gram-negative bacteria and is unable to penetrate the outer membrane barrier. In an effort to determine whether vancomycin and other antibiotics effective against Gram-positive bacteria could, through modification, be rendered effective against Gram-negative bacteria, we discovered that the covalent attachment of a single arginine to vancomycin yielded conjugates with order-of-magnitude improvements in activity against Gram-negative bacteria, including pathogenic E. coli. The vancomycin-arginine conjugate (V-R) exhibited efficacy against actively growing bacteria, induced the loss of rod cellular morphology, and resulted in the intracellular accumulation of peptidoglycan precursors, all consistent with cell-wall synthesis disruption as its mechanism of action. Membrane permeabilization studies demonstrated an enhanced outer membrane permeability of V-R as compared with vancomycin. The conjugate exhibited no mammalian cell toxicity or hemolytic activity in MTT and hemolysis assays. Our study introduces a new vancomycin derivative effective against Gram-negative bacteria and underscores the broader potential of generating new antibiotics through combined mode-of-action and synthesis-informed design studies.

    View details for DOI 10.1021/acschembio.9b00565

    View details for PubMedID 31479234

  • Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenic Escherichia coli to bladder epithelial cells. Proceedings of the National Academy of Sciences of the United States of America Hollenbeck, E. C., Antonoplis, A., Chai, C., Thongsomboon, W., Fuller, G. G., Cegelski, L. 2018


    Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infections, employing numerous molecular strategies to contribute to adhesion, colonization, and persistence in the bladder niche. Identifying strategies to prevent adhesion and colonization is a promising approach to inhibit bacterial pathogenesis and to help preserve the efficacy of available antibiotics. This approach requires an improved understanding of the molecular determinants of adhesion to the bladder urothelium. We designed experiments using a custom-built live cell monolayer rheometer (LCMR) to quantitatively measure individual and combined contributions of bacterial cell surface structures [type 1 pili, curli, and phosphoethanolamine (pEtN) cellulose] to bladder cell adhesion. Using the UPEC strain UTI89, isogenic mutants, and controlled conditions for the differential production of cell surface structures, we discovered that curli can promote stronger adhesive interactions with bladder cells than type 1 pili. Moreover, the coproduction of curli and pEtN cellulose enhanced adhesion. The LCMR enables the evaluation of adhesion under high-shear conditions to reveal this role for pEtN cellulose which escaped detection using conventional tissue culture adhesion assays. Together with complementary biochemical experiments, the results support a model wherein cellulose serves a mortar-like function to promote curli association with and around the bacterial cell surface, resulting in increased bacterial adhesion strength at the bladder cell surface.

    View details for PubMedID 30232265

  • Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose SCIENCE Thongsomboon, W., Serra, D. O., Possling, A., Hadjineophytou, C., Hengge, R., Cegelski, L. 2018; 359 (6373): 334–38


    Cellulose is a major contributor to the chemical and mechanical properties of plants and assumes structural roles in bacterial communities termed biofilms. We find that Escherichia coli produces chemically modified cellulose that is required for extracellular matrix assembly and biofilm architecture. Solid-state nuclear magnetic resonance spectroscopy of the intact and insoluble material elucidates the zwitterionic phosphoethanolamine modification that had evaded detection by conventional methods. Installation of the phosphoethanolamine group requires BcsG, a proposed phosphoethanolamine transferase, with biofilm-promoting cyclic diguanylate monophosphate input through a BcsE-BcsF-BcsG transmembrane signaling pathway. The bcsEFG operon is present in many bacteria, including Salmonella species, that also produce the modified cellulose. The discovery of phosphoethanolamine cellulose and the genetic and molecular basis for its production offers opportunities to modulate its production in bacteria and inspires efforts to biosynthetically engineer alternatively modified cellulosic materials.

    View details for PubMedID 29348238

  • A dual function antibiotic-transporter conjugate exhibits superior activity in sterilizing MRSA biofilms and killing persister cells. Journal of the American Chemical Society Antonoplis, A. n., Zang, X. n., Huttner, M. A., Chong, K. n., Lee, Y. B., Co, J. Y., Amieva, M. n., Kline, K. n., Wender, P. A., Cegelski, L. n. 2018


    New strategies are urgently needed to target MRSA, a major global health problem and the leading cause of mortality from antibiotic-resistant infections in many countries. Here we report a general approach to this problem exemplified by the design and synthesis of a vancomycin-D-octaarginine conjugate (V-r8) and investigation of its efficacy in addressing antibiotic-insensitive bacterial populations. V-r8 eradicated MRSA biofilm and persister cells in vitro, outperforming vancomycin by orders of magnitude. It also eliminated 97% of biofilm-associated MRSA in a murine wound infection model and displayed no acute dermal toxicity. This new dual function conjugate displays enhanced cellular accumulation and membrane perturba-tion as compared to vancomycin. Based on its rapid and potent activity against biofilm and persister cells, V-r8 is a promis-ing agent against clinical MRSA infections.

    View details for PubMedID 30388366

  • Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR. Philosophical transactions of the Royal Society of London. Series B, Biological sciences Romaniuk, J. A., Cegelski, L. 2015; 370 (1679)


    The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms.

    View details for DOI 10.1098/rstb.2015.0024

    View details for PubMedID 26370936

  • Detection of intact vancomycin-arginine as the active antibacterial conjugate in E. coli by whole-cell solid-state NMR. RSC medicinal chemistry Werby, S. H., Brčić, J., Chosy, M. B., Sun, J., Rendell, J. T., Neville, L. F., Wender, P. A., Cegelski, L. 2023; 14 (6): 1192-1198


    The introduction of new and improved antibacterial agents based on facile synthetic modifications of existing antibiotics represents a promising strategy to deliver urgently needed antibacterial candidates to treat multi-drug resistant bacterial infections. Using this strategy, vancomycin was transformed into a highly active agent against antibiotic-resistant Gram-negative organisms in vitro and in vivo through the addition of a single arginine to yield vancomycin-arginine (V-R). Here, we report detection of the accumulation of V-R in E. coli by whole-cell solid-state NMR using 15N-labeled V-R. 15N CPMAS NMR revealed that the conjugate remained fully amidated without loss of arginine, demonstrating that intact V-R represents the active antibacterial agent. Furthermore, C{N}REDOR NMR in whole cells with all carbons at natural abundance 13C levels exhibited the sensitivity and selectivity to detect the directly bonded 13C-15N pairs of V-R within E. coli cells. Thus, we also present an effective methodology to directly detect and evaluate active drug agents and their accumulation within bacteria without the need for potentially perturbative cell lysis and analysis protocols.

    View details for DOI 10.1039/d3md00173c

    View details for PubMedID 37360389

    View details for PubMedCentralID PMC10285746

  • Nordihydroguaiaretic Acid (NDGA) Inhibits CsgA Polymerization, Bacterial Amyloid Biogenesis, and Biofilm Formation. Chembiochem : a European journal of chemical biology Visser, J. A., Yager, D., Chambers, S. A., Lim, J. Y., Cao, X., Cegelski, L. 2023: e202300266


    Escherichia coli and other Enterobacteriaceae thrive in robust biofilm communities through the coproduction of curli amyloid fibers and phosphoethanolamine cellulose. Curli promote adhesion to abiotic surfaces and plant and human host tissues and are associated with pathogenesis in urinary tract infection and foodborne illness. As amyloid, curli production in the host has also been implicated in the pathogenesis of neurodegenerative diseases. We report that the natural product nordihydroguaiaretic acid (NDGA) is effective as a curlicide in E. coli. NDGA prevents CsgA polymerization in vitro in a dose-dependent manner. NDGA selectively inhibits cellassociated curli assembly in E. coli and inhibits biofilm formation among uropathogenic E. coli in a curli-specific manner. More broadly, our work emphasizes the ability to evaluate and identify bioactive amyloid assembly inhibitors using the powerful gene-directed amyloid biogenesis machinery in E. coli.

    View details for DOI 10.1002/cbic.202300266

    View details for PubMedID 37195016

  • Catching Threads in Bacterial Cell Walls ACS CENTRAL SCIENCE Kallem, T., Cegelski, L. 2022: 1376-1379

    View details for DOI 10.1021/acscentsci.2c01070

    View details for Web of Science ID 000870256300001

    View details for PubMedID 36313163

    View details for PubMedCentralID PMC9615111

  • PITing it forward: A new link in the journey of uropathogenic E.coli in the urothelium. Cell reports Joshi, C. S., Cegelski, L., Mysorekar, I. U. 2022; 39 (4): 110758


    Urinary tract infections (UTIs) are a cause for alarm given the high rates of treatment failure. In a recent issue of Cell Reports, Pang etal. uncovered dueling molecular machinery at the host-pathogen interface in response to phosphate that points to new anti-infective strategies against UTIs.

    View details for DOI 10.1016/j.celrep.2022.110758

    View details for PubMedID 35476986

  • Chemical and Molecular Composition of the Chrysalis Reveals Common Chitin-rich Structural Framework for Monarchs and Swallowtails. Journal of molecular biology Goularte, N. F., Kallem, T., Cegelski, L. 1800: 167456


    The metamorphosis of a caterpillar into a butterfly is an awe-inspiring example of how extraordinary functions are made possible through specific chemistry in nature's complex systems. The chrysalis exoskeleton is revealed and shed as a caterpillar transitions to butterfly form. We employed solid-state NMR to evaluate the chemical composition and types of biomolecules in the chrysalides from which Monarch and Swallowtail butterflies emerged. The chrysalis composition was remarkably similar between Monarch and Swallowtail. Chitin is the major polysaccharide component, present together with proteins and catechols or catechol-type linkages in each chrysalis. The high chitin content is comparable to the highest chitin-containing insect exoskeletons. Proteomics analysis of associated soluble proteins indicated the presence of chitinases that could be involved in synthesis and remodeling of the chrysalis as well as key cuticular proteins which play a role in the structural integrity of the chrysalis. The nearly identical 13C CPMAS NMR spectra of each chrysalis and similar structural proteins supports the presence of underlying design principles integrating chitin and protein partners to elaborate the chrysalis.

    View details for DOI 10.1016/j.jmb.2022.167456

    View details for PubMedID 35045329

  • Molecular organization of the E. coli cellulose synthase macrocomplex. Nature structural & molecular biology Acheson, J. F., Ho, R., Goularte, N. F., Cegelski, L., Zimmer, J. 2021; 28 (3): 310–18


    Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue adhesion. The E. coli pEtN cellulose biosynthesis machinery contains the catalytic BcsA-B complex that synthesizes and secretes cellulose, in addition to five other subunits. The membrane-anchored periplasmic BcsG subunit catalyzes pEtN modification. Here we present the structure of the roughly 1MDa E. coli Bcs complex, consisting of one BcsA enzyme associated with six copies of BcsB, determined by single-particle cryo-electron microscopy. BcsB homo-oligomerizes primarily through interactions between its carbohydrate-binding domains as well as intermolecular beta-sheet formation. The BcsB hexamer creates a half spiral whose open side accommodates two BcsG subunits, directly adjacent to BcsA's periplasmic channel exit. The cytosolic BcsE and BcsQ subunits associate with BcsA's regulatory PilZ domain. The macrocomplex is a fascinating example of cellulose synthase specification.

    View details for DOI 10.1038/s41594-021-00569-7

    View details for PubMedID 33712813

  • Identification of a novel pyruvyltransferase using 13C solid-state NMR to analyze rhizobial exopolysaccharides. Journal of bacteriology Wells, D. H., Goularte, N. F., Barnett, M. J., Cegelski, L., Long, S. R. 2021: JB0040321


    The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPS), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially enable it to adapt to a changing environment. Succinoglycan is essential for invasion of plant hosts, and thus for formation of nitrogen-fixing root nodules. Galactoglucan is critical for population-based behaviors such as swarming and biofilm formation, and can facilitate invasion in the absence of succinoglycan on some host plants. Biosynthesis of galactoglucan is not as completely understood as that of succinoglycan. We devised a pipeline to: identify putative pyruvyltransferase and acetyltransferase genes; construct genomic deletions in strains engineered to produce either succinoglycan or galactoglucan; and analyze EPS from mutant bacterial strains. EPS samples were examined by 13C cross-polarization magic-angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). CPMAS NMR is uniquely suited to defining chemical composition in complex samples and enable detection and quantification of distinct EPS functional groups. Galactoglucan was isolated from mutant strains, with deletions in five candidate acyl/acetyltransferase genes (exoZ, exoH, SMb20810, SMb21188, SMa1016) and a putative pyruvyltransferase (wgaE or SMb21322). Most samples were similar in composition to wild-type EPSII by CPMAS NMR analysis. However, galactoglucan produced from a strain lacking wgaE exhibited a significant reduction in pyruvylation. Pyruvylation was restored through ectopic expression of plasmid-encoded wgaE. Our work has thus identified WgaE as a galactoglucan pyruvyltransferase. This exemplifies how the systematic combination of genetic analyses and solid-state NMR detection is a rapid means to identify genes responsible for modification of rhizobial exopolysaccharides. IMPORTANCE Nitrogen-fixing bacteria are crucial for geochemical cycles and global nitrogen nutrition. Symbioses between legumes and rhizobial bacteria establish root nodules, where bacteria convert dinitrogen to ammonia for plant utilization. Secreted exopolysaccharides (EPS) produced by Sinorhizobium meliloti (succinoglycan and galactoglucan) play important roles in soil and plant environments. Biosynthesis of galactoglucan is not as well characterized as succinoglycan. We employed solid-state nuclear magnetic resonance (NMR) to examine intact EPS from wild type and mutant S. meliloti strains. NMR analysis of EPS isolated from a wgaE gene mutant revealed a novel pyruvyltransferase that modifies galactoglucan. Few EPS pyruvyltransferases have been characterized. Our work provides insight into biosynthesis of an important S. meliloti EPS and expands knowledge of enzymes that modify polysaccharides.

    View details for DOI 10.1128/JB.00403-21

    View details for PubMedID 34606371

  • Mechanochemical synthesis of an elusive fluorinated polyacetylene. Nature chemistry Boswell, B. R., Mansson, C. M., Cox, J. M., Jin, Z., Romaniuk, J. A., Lindquist, K. P., Cegelski, L., Xia, Y., Lopez, S. A., Burns, N. Z. 2020


    Polymer mechanochemistry has traditionally been employed to study the effects of mechanical force on chemical bonds within a polymer backbone or to generate force-responsive materials. It is under-exploited for the scalable synthesis of wholly new materials by chemically transforming the polymers, especially products inaccessible by other means. Here we utilize polymer mechanochemistry to synthesize a fluorinated polyacetylene, a long-sought-after air-stable polyacetylene that has eluded synthesis by conventional means. We construct the monomer in four chemical steps on gram scale, which involves a rapid incorporation of fluorine atoms in an exotic photochemical cascade whose mechanism and exquisite stereoselectivity were informed by computation. After polymerization, force activation by ultrasonication produces a gold-coloured, semiconducting fluoropolymer. This work demonstrates that polymer mechanochemistry is a valuable synthetic tool for accessing materials on a preparative scale.

    View details for DOI 10.1038/s41557-020-00608-8

    View details for PubMedID 33349696

  • Variation in the ratio of curli and phosphoethanolamine cellulose associated with biofilm architecture and properties. Biopolymers Jeffries, J., Thongsomboon, W., Visser, J. A., Enriquez, K., Yager, D., Cegelski, L. 2020: e23395


    Bacterial biofilms are communities of bacteria entangled in a self-produced extracellular matrix (ECM). Escherichia coli direct the assembly of two insoluble biopolymers, curli amyloid fibers, and phosphoethanolamine (pEtN) cellulose, to build remarkable biofilm architectures. Intense curiosity surrounds how bacteria harness these amyloid-polysaccharide composites to build biofilms, and how these biopolymers function to benefit bacterial communities. Defining ECM composition involving insoluble polymeric assemblies poses unique challenges to analysis and, thus, to comparing strains with quantitative ECM molecular correlates. In this work, we present results from a sum-of-the-parts 13 C solid-state nuclear magnetic resonance (NMR) analysis to define the curli-to-pEtN cellulose ratio in the isolated ECM of the E. coli laboratory K12 strain, AR3110. We compare and contrast the compositional analysis and comprehensive biofilm phenotypes for AR3110 and a well-studied clinical isolate, UTI89. The ECM isolated from AR3110 contains approximately twice the amount of pEtN cellulose relative to curli content as UTI89, revealing plasticity in matrix assembly principles among strains. The two parent strains and a panel of relevant gene mutants were investigated in three biofilm models, examining: (a) macrocolonies on agar, (b) pellicles at the liquid-air interface, and (c) biomass accumulation on plastic. We describe the influence of curli, cellulose, and the pEtN modification on biofilm phenotypes with power in the direct comparison of these strains. The results suggest that curli more strongly influence adhesion, while pEtN cellulose drives cohesion. Their individual and combined influence depends on both the biofilm modality (agar, pellicle, or plastic-associated) and the strain itself.

    View details for DOI 10.1002/bip.23395

    View details for PubMedID 32894594

  • Bicyclohexene-peri-naphthalenes: Scalable Synthesis, Diverse Functionalization, Efficient Polymerization, and Facile Mechanoactivation of Their Polymers. Journal of the American Chemical Society Yang, J., Horst, M., Werby, S. H., Cegelski, L., Burns, N. Z., Xia, Y. 2020; 142 (34): 14619–26


    Pursuing polymers that can transform from a nonconjugated to a conjugated state under mechanical stress to significantly change their properties, we developed a new generation of ladder-type mechanophore monomers, bicyclo[2.2.0]hex-5-ene-peri-naphthalene (BCH-Naph), that can be directly and efficiently polymerized by ring-opening metathesis polymerization (ROMP). BCH-Naphs can be synthesized in multigram quantities and functionalized with a wide range of electron-rich and electron-poor substituents, allowing tuning of the optoelectronic and physical properties of mechanically generated conjugated polymers. Efficient ROMP of BCH-Naphs yielded ultrahigh molecular weight polymechanophores with controlled MWs and low dispersity. The resulting poly(BCH-Naph)s can be mechanically activated into conjugated polymers using ultrasonication, grinding, and even simple stirring of the dilute solutions, leading to changes in absorption and fluorescence. Poly(BCH-Naph)s represent an attractive polymechanophore system to explore multifaceted mechanical response in solution and solid states, owing to the synthetic scalability, functional diversity, efficient polymerization, and facile mechanoactivation.

    View details for DOI 10.1021/jacs.0c06454

    View details for PubMedID 32786795

  • Evaluation of Phosphoethanolamine Cellulose Production among Bacterial Communities using Congo Red Fluorescence. Journal of bacteriology Thongsomboon, W., Werby, S. H., Cegelski, L. 2020


    Bacterial biofilms are surface-associated communities of bacterial cells enmeshed in an extracellular matrix (ECM). The biofilm lifestyle results in physiological heterogeneity across the community, promotes persistence, and protects cells from external insults such as antibiotic treatment. E. coli was recently discovered to produce a chemically modified form of cellulose, phosphoethanolamine (pEtN) cellulose, contributing to the formation of its extracellular matrix and elaboration of its hallmark wrinkled macrocolony architectures. Both pEtN cellulose and unmodified cellulose bind dyes such as Calcofluor White and Congo red (CR). Here, we present the use of CR fluorescence to distinguish between pEtN cellulose and unmodified cellulose producers. We demonstrate the utility of this tool in the evaluation of a uropathogenic E. coli clinical isolate that appeared to produce curli and a cellulosic component, but did not exhibit macrocolony wrinkling. We determined that lack of macrocolony wrinkling was attributed to a single nucleotide mutation and introduction of a stop codon in bcsG, abrogating production of BcsG, the pEtN transferase. Thus, this work underscores the important contribution of the pEtN cellulose modification to the E. coli agar-based macrocolony wrinkling phenotype and introduces a facile approach to distinguish between modified and unmodified cellulose.IMPORTANCE E. coli produce amyloid fibers termed curli and a cellulosic component to assemble biofilm communities. Cellulose is the most abundant biopolymer on Earth and we recently discovered that the cellulosic component in E. coli biofilms was not standard cellulose, but a newly identified cellulosic polymer - phosphoethanolamine cellulose. Studies involving the biological and functional impact of the cellulose modification among E. coli and other organisms are just beginning. Convenient methods for distinguishing pEtN cellulose from unmodified cellulose in E. coli and estimating production are needed to facilitate further research. Dissecting the balance of pEtN cellulose and curli production by E. coli commensal strains and clinical isolates will improve our understanding of the host microbiome and factors contributing to bacterial pathogenesis.

    View details for DOI 10.1128/JB.00030-20

    View details for PubMedID 32312746

  • Mechanical and microstructural insights of Vibrio cholerae and Escherichia coli dual-species biofilm at the air-liquid interface. Colloids and surfaces. B, Biointerfaces Abriat, C., Enriquez, K., Virgilio, N., Cegelski, L., Fuller, G. G., Daigle, F., Heuzey, M. 2020; 188: 110786


    Biofilm is the dominant microbial form found in nature, in which bacterial species are embedded in a self-produced extracellular matrix (ECM). These complex microbial communities are responsible for several infections when they involve multispecies pathogenic bacteria. In previous studies, interfacial rheology proved to be a unique quantitative technique to follow in real-time the biofilm formation at the air-liquid interface. In this work, we studied a model system composed of two bacteria pathogenic capable of forming a pellicle biofilm, V. cholerae and E. coli. We used an integrated approach by combining a real-time quantitative analysis of the biofilm rheological properties, with the investigation of major matrix components and the pellicle microstructure. The results highlight the competition for the interface between the two species, driven by the biofilm formation growth rate. In the dual-species biofilm, the viscoelastic properties were dominated by V. cholera, which formed a mature biofilm 18 h faster than E. coli. The microstructure of the dual-species biofilm revealed a similar morphology to V. cholerae alone when both bacteria were initially added at the same amount. The analysis of some major ECM components showed that E. coli was not able to produce curli in the presence of V. cholerae, unless enough time was given for E. coli to colonize the air-liquid interface first. E. coli secreted phosphoethanolamine (pEtN) cellulose in the dual-species biofilm, but did not form a filamentous structure. Our pathogenic model system demonstrated the importance of the biofilm growth rate for multispecies biofilm composition at the air-liquid interface.

    View details for DOI 10.1016/j.colsurfb.2020.110786

    View details for PubMedID 31954270

  • Design and Implementation of a Six-Session CURE Module Using Biofilms to Explore the Chemistry-Biology Interface JOURNAL OF CHEMICAL EDUCATION Werby, S., Cegelski, L. 2019; 96 (9): 2050–54
  • Benzoladderene Mechanophores: Synthesis, Polymerization, and Mechanochemical Transformation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yang, J., Horst, M., Romaniuk, J. H., Jin, Z., Cegelski, L., Xia, Y. 2019; 141 (16): 6479–83
  • Benzoladderene Mechanophores: Synthesis, Polymerization, and Mechanochemical Transformation. Journal of the American Chemical Society Yang, J., Horst, M., Romaniuk, J. A., Jin, Z., Cegelski, L., Xia, Y. 2019


    We have previously reported a polymechanophore system, polyladderene, which underwent dramatic bond rearrangement in response to mechanical force to yield semiconducting polyacetylene. Herein, we report the scalable synthesis of benzoladderenes as new mechanophore monomers. Ring-opening metathesis polymerization of benzoladderenes yielded homopolymers and block copolymers with controlled molecular weights and low dispersity. The resulting nonconjugated poly(benzoladderene) was mechanochemically transformed into conjugated poly( o-phenylene-hexatrienylene) by sonication, with degrees of transformation up to 40-45%. These benzoladderenes and their resulting polymers are easier to synthesize than the polyladderene system and allow mechanochemical generation of conjugated polymersbeyond polyacetylene.

    View details for PubMedID 30969109

  • Integration of electron microscopy and solid-state NMR analysis for new views and compositional parameters of Aspergillus fumigatus biofilms MEDICAL MYCOLOGY Reichhardt, C., Joubert, L., Clemons, K. V., Stevens, D. A., Cegelski, L. 2019; 57: S239–S244

    View details for DOI 10.1093/mmy/myy140

    View details for Web of Science ID 000463726800020

  • Integration of electron microscopy and solid-state NMR analysis for new views and compositional parameters of Aspergillus fumigatus biofilms. Medical mycology Reichhardt, C., Joubert, L., Clemons, K. V., Stevens, D. A., Cegelski, L. 2019; 57 (Supplement_2): S239–S244


    The general ability and tendency of bacteria and fungi to assemble into bacterial communities, termed biofilms, poses unique challenges to the treatment of human infections. Fungal biofilms, in particular, are associated with enhanced virulence in vivo and decreased sensitivity to antifungals. Much attention has been given to the complex cell wall structures in fungal organisms, yet beyond the cell surface, Aspergillus fumigatus and other fungi assemble a self-secreted extracellular matrix that is the hallmark of the biofilm lifestyle, protecting and changing the environment of resident members. Elucidation of the chemical and molecular detail of the extracellular matrix is crucial to understanding how its structure contributes to persistence and antifungal resistance in the host. We present a summary of integrated analyses of A. fumigatus biofilm architecture, including hyphae and the extracellular matrix, by scanning electron microscopy and A. fumigatus matrix composition by new top-down solid-state NMR approaches coupled with biochemical analysis. This combined methodology will be invaluable in formulating quantitative and chemical comparisons of A. fumigatus isolates that differ in virulence and are more or less resistant to antifungals. Ultimately, knowledge of the chemical and molecular requirements for matrix formation and function will drive the identification and development of new strategies to interfere with biofilm formation and virulence.

    View details for PubMedID 30816969

  • Spectral comparisons of mammalian cells and intact organelles by solid-state NMR JOURNAL OF STRUCTURAL BIOLOGY Werby, S. H., Cegelski, L. 2019; 206 (1): 49–54
  • Respiratory Heterogeneity Shapes Biofilm Formation and Host Colonization in Uropathogenic Escherichia coli MBIO Beebout, C. J., Eberly, A. R., Werby, S. H., Reasoner, S. A., Brannon, J. R., De, S., Fitzgerald, M. J., Huggins, M. M., Clayton, D. B., Cegelski, L., Hadjifrangiskou, M. 2019; 10 (2)
  • Carbon compositional analysis of hydrogel contact lenses by solid-state NMR spectroscopy. Solid state nuclear magnetic resonance Rabiah, N. I., Romaniuk, J. A., Fuller, G. G., Scales, C. W., Cegelski, L. n. 2019; 102: 47–52


    Contact lenses are worn by over 140 million people each year and tremendous research and development efforts contribute to the identification and selection of hydrogel components and production protocols to yield lenses optimized for chemical and physiological properties, eye health and comfort. The final molecular composition and extent of incorporation of different components in contact lenses is routinely estimated after lens production through the analysis of the soluble components that were not included in the lens, i.e. remaining starting materials. Examination of composition in the actual intact materials is always valued and can reveal details that are missed by only examining the non-incorporated components, for example identifying chemical changes to components in lenses during the production process. Solid-state nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for the direct compositional analysis of insoluble and heterogeneous materials and is also uniquely suited to determining parameters of architecture in contact lenses. We utilized 13C cross-polarization magic angle spinning (CPMAS) NMR to examine and compare the carbon composition of soft contact lenses. 13C NMR spectra of individual polymer components enabled the determination of the approximate molecular carbon contributions of major lens components. Comparisons of the conventional etafilcon A hydrogel (1 Day Acuvue MOIST) lenses and silicone hydrogel lenses (Acuvue Oasys, Dailies Total 1, Clariti 1 Day, Biofinity, and Pure Vision) revealed major spectral differences, with considerable variation even among different silicone hydrogel lenses. The solid-state NMR approach provides a direct spectral reporting of carbon types in the hydrogel lens itself. This approach represents a valuable complementary analysis to benefit contact lens research and development and could be extended to isotopically labeled hydrogel lenses to map proximities and architecture between hydrogel components.

    View details for DOI 10.1016/j.ssnmr.2019.07.003

    View details for PubMedID 31376631

  • Unraveling Escherichia coli's Cloak: Identification of Phosphoethanolamine Cellulose, Its Functions, and Applications. Microbiology insights Jeffries, J. n., Fuller, G. G., Cegelski, L. n. 2019; 12: 1178636119865234


    Bacterial biofilms are complex, multicellular communities made up of bacteria enmeshed in a self-produced extracellular matrix (ECM) that protects against environmental stress. The ECM often comprises insoluble components, which complicates the study of biofilm composition, structure, and function. Wrinkled, agar-grown Escherichia coli biofilms require 2 insoluble macromolecules: curli amyloid fibers and cellulosic polymers. We quantified these components with solid-state nuclear magnetic resonance (NMR) and determined that curli contributed 85% of the isolated uropathogenic E coli ECM dry mass. The remaining 15% was cellulosic, but, surprisingly, was not ordinary cellulose. We tracked the identity of the unanticipated peak in the 13C NMR spectrum of the cellulosic component and discovered that E coli secrete phosphoethanolamine (pEtN)-modified cellulose. Cellulose is the most abundant biopolymer on the planet, and this marked the first identification of a naturally, chemically modified cellulose. To investigate potential roles of pEtN cellulose, we customized a newly designed live-cell monolayer rheometer and demonstrated that pEtN cellulose facilitated E coli attachment to bladder epithelial cells and acted as a glue, keeping curli cell associated. The discovery of pEtN cellulose opens questions regarding its biological function(s) and provides opportunities in materials science to explore this newly discovered biopolymer.

    View details for DOI 10.1177/1178636119865234

    View details for PubMedID 31431800

    View details for PubMedCentralID PMC6685106

  • Respiratory Heterogeneity Shapes Biofilm Formation and Host Colonization in Uropathogenic Escherichia coli. mBio Beebout, C. J., Eberly, A. R., Werby, S. H., Reasoner, S. A., Brannon, J. R., De, S., Fitzgerald, M. J., Huggins, M. M., Clayton, D. B., Cegelski, L., Hadjifrangiskou, M. 2019; 10 (2)


    Biofilms are multicellular bacterial communities encased in a self-secreted extracellular matrix comprised of polysaccharides, proteinaceous fibers, and DNA. Organization of these components lends spatial organization to the biofilm community such that biofilm residents can benefit from the production of common goods while being protected from exogenous insults. Spatial organization is driven by the presence of chemical gradients, such as oxygen. Here we show that two quinol oxidases found in Escherichia coli and other bacteria organize along the biofilm oxygen gradient and that this spatially coordinated expression controls architectural integrity. Cytochrome bd, a high-affinity quinol oxidase required for aerobic respiration under hypoxic conditions, is the most abundantly expressed respiratory complex in the biofilm community. Depletion of the cytochrome bd-expressing subpopulation compromises biofilm complexity by reducing the abundance of secreted extracellular matrix as well as increasing cellular sensitivity to exogenous stresses. Interrogation of the distribution of quinol oxidases in the planktonic state revealed that 15% of the population expresses cytochrome bd at atmospheric oxygen concentration, and this population dominates during acute urinary tract infection. These data point toward a bet-hedging mechanism in which heterogeneous expression of respiratory complexes ensures respiratory plasticity of E. coli across diverse host niches.IMPORTANCE Biofilms are multicellular bacterial communities encased in a self-secreted extracellular matrix comprised of polysaccharides, proteinaceous fibers, and DNA. Organization of these components lends spatial organization in the biofilm community. Here we demonstrate that oxygen gradients in uropathogenic Escherichia coli (UPEC) biofilms lead to spatially distinct expression programs for quinol oxidases-components of the terminal electron transport chain. Our studies reveal that the cytochrome bd-expressing subpopulation is critical for biofilm development and matrix production. In addition, we show that quinol oxidases are heterogeneously expressed in planktonic populations and that this respiratory heterogeneity provides a fitness advantage during infection. These studies define the contributions of quinol oxidases to biofilm physiology and suggest the presence of respiratory bet-hedging behavior in UPEC.

    View details for PubMedID 30940709

  • Functional Specialization in Vibrio cholerae Diguanylate Cyclases: Distinct Modes of Motility Suppression and c-di-GMP Production. mBio Zamorano-Sánchez, D. n., Xian, W. n., Lee, C. K., Salinas, M. n., Thongsomboon, W. n., Cegelski, L. n., Wong, G. C., Yildiz, F. H. 2019; 10 (2)


    Vibrio cholerae biofilm formation and associated motility suppression are correlated with increased concentrations of cyclic diguanylate monophosphate (c-di-GMP), which are in turn driven by increased levels and/or activity of diguanylate cyclases (DGCs). To further our understanding of how c-di-GMP modulators in V. cholerae individually and collectively influence motility with cellular resolution, we determined how DGCs CdgD and CdgH impact intracellular c-di-GMP levels, motility, and biofilm formation. Our results indicated that CdgH strongly influences swim speed distributions; cells in which cdgH was deleted had higher average swim speeds than wild-type cells. Furthermore, our results suggest that CdgD, rather than CdgH, is the dominant DGC responsible for postattachment c-di-GMP production in biofilms. Lipopolysaccharide (LPS) biosynthesis genes were found to be extragenic bypass suppressors of the motility phenotypes of strains ΔcdgD and ΔcdgH We compared the motility regulation mechanism of the DGCs with that of Gmd, an LPS O-antigen biosynthesis protein, and discovered that comodulation of c-di-GMP levels by these motility effectors can be positively or negatively cooperative rather than simply additive. Taken together, these results suggest that different environmental and metabolic inputs orchestrate DGC responses of V. cholerae via c-di-GMP production and motility modulation.IMPORTANCE Cyclic diguanylate monophosphate (c-di-GMP) is a broadly conserved bacterial signaling molecule that affects motility, biofilm formation, and virulence. Although it has been known that high intracellular concentrations of c-di-GMP correlate with motility suppression and biofilm formation, how the 53 predicted c-di-GMP modulators in Vibrio cholerae collectively influence motility is not understood in detail. Here we used a combination of plate assays and single-cell tracking methods to correlate motility and biofilm formation outcomes with specific enzymes involved in c-di-GMP synthesis in Vibrio cholerae, the causative agent of the disease cholera.

    View details for PubMedID 31015332

  • Synthesis and Mechanochemical Activation of Ladderene-Norbornene Block Copolymers. Journal of the American Chemical Society Su, J. K., Feist, J. D., Yang, J., Mercer, J. A., Romaniuk, J. A., Chen, Z., Cegelski, L., Burns, N. Z., Xia, Y. 2018; 140 (39): 12388–91


    We have recently reported a polymechanophore system, polyladderene (PLDE), which dramatically transforms into polyacetylene (PA) upon mechanical stimulation. Herein, we optimized conditions to synthesize unprecedented block copolymers (BCPs) containing a force-responsive block by sequential ring-opening metathesis polymerization of different norbornenes and bromoladderene. Successful extension from PLDE to other blocks required careful timing and low temperatures to preserve the reactivity of the PLDE-appended catalyst. The PLDE-containing BCPs were sonochemically activated into visually soluble PA with a maximum absorption lambda ≥ 600 nm and unique absorption patterns corresponding to noncontinuous activation of ladderene units. Access to polymechanophore BCPs paves the way for new stress-responsive materials with solution and solid state self-assembly behaviors and incorporation of polymechanophores into other materials.

    View details for PubMedID 30229652

  • Peptidoglycan and Teichoic Acid Levels and Alterations in Staphylococcus aureus by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance BIOCHEMISTRY Romaniuk, J. H., Cegelski, L. 2018; 57 (26): 3966–75


    Gram-positive bacteria surround themselves with a multilayered macromolecular cell wall that is essential to cell survival and serves as a major target for antibiotics. The cell wall of Staphylococcus aureus is composed of two major structural components, peptidoglycan (PG) and wall teichoic acid (WTA), together creating a heterogeneous and insoluble matrix that poses a challenge to quantitative compositional analysis. Here, we present 13C cross polarization magic angle spinning solid-state nuclear magnetic resonance (NMR) spectra of intact cell walls, purified PG, and purified WTA. The spectra reveal the clear molecular differences in the two polymers and enable quantification of PG and WTA in isolated cell walls, an attractive alternative to estimating teichoic acid content from a phosphate analysis of completely pyrolyzed cell walls. Furthermore, we discovered that unique PG and WTA spectral signatures could be identified in whole-cell NMR spectra and used to compare PG and WTA levels among intact bacterial cell samples. The distinguishing whole-cell 13C NMR contributions associated with PG include the GlcNAc-MurNAc sugar carbons and glycyl α-carbons. WTA contributes carbons from the phosphoribitol backbone. Distinguishing 15N spectral signatures include glycyl amide nitrogens in PG and the esterified d-alanyl amine nitrogens in WTA. 13C NMR analysis was performed with samples at natural abundance and included 10 whole-cell sample comparisons. Changes consistent with altered PG and WTA content were detected in whole-cell spectra of bacteria harvested at different growth times and in cells treated with tunicamycin. This use of whole-cell NMR provides quantitative parameters of composition in the context of whole-cell activity.

    View details for PubMedID 29806458

  • REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment ACS CENTRAL SCIENCE Yang, H., Staveness, D., Ryckbosch, S. M., Axtman, A. D., Loy, B. A., Barnes, A. B., Pande, V. S., Schaefer, J., Wender, P. A., Cegelski, L. 2018; 4 (1): 89–96


    Bryostatin 1 (henceforth bryostatin) is in clinical trials for the treatment of Alzheimer's disease and for HIV/AIDS eradication. It is also a preclinical lead for cancer immunotherapy and other therapeutic indications. Yet nothing is known about the conformation of bryostatin bound to its protein kinase C (PKC) target in a membrane microenvironment. As a result, efforts to design more efficacious, better tolerated, or more synthetically accessible ligands have been limited to structures that do not include PKC or membrane effects known to influence PKC-ligand binding. This problem extends more generally to many membrane-associated proteins in the human proteome. Here, we use rotational-echo double-resonance (REDOR) solid-state NMR to determine the conformations of PKC modulators bound to the PKCδ-C1b domain in the presence of phospholipid vesicles. The conformationally limited PKC modulator phorbol diacetate (PDAc) is used as an initial test substrate. While unanticipated partitioning of PDAc between an immobilized protein-bound state and a mobile state in the phospholipid assembly was observed, a single conformation in the bound state was identified. In striking contrast, a bryostatin analogue (bryolog) was found to exist exclusively in a protein-bound state, but adopts a distribution of conformations as defined by three independent distance measurements. The detection of multiple PKCδ-C1b-bound bryolog conformers in a functionally relevant phospholipid complex reveals the inherent dynamic nature of cellular systems that is not captured with single-conformation static structures. These results indicate that binding, selectivity, and function of PKC modulators, as well as the design of new modulators, are best addressed using a dynamic multistate model, an analysis potentially applicable to other membrane-associated proteins.

    View details for PubMedID 29392180

    View details for PubMedCentralID PMC5785774

  • The Congo red derivative FSB binds to curli amyloid fibers and specifically stains curliated E. coli. PloS one Reichhardt, C., Cegelski, L. 2018; 13 (8): e0203226


    The Congo red derivative (E,E)-1-fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy) styrylbenzene (FSB) specifically stains the functional amyloid curli in Escherichia coli biofilms. FSB binds to curli with similar affinity as Congo red, yet exhibits much greater fluorescence upon binding to curli as compared to Congo red and does not exhibit undesired binding to the cellulosic component of the biofilm. Thus, FSB presents a powerful tool to identify and visualize curli in E. coli biofilms and also enables new biophysical investigations of curli.

    View details for PubMedID 30161215

  • Whole-Cell Detection of C-P Bonds in Bacteria BIOCHEMISTRY Bartlett, C., Bansal, S., Burnett, A., Suits, M. D., Schaefer, J., Cegelski, L., Horsman, G. P., Weadge, J. T. 2017; 56 (44): 5870–73


    Naturally produced molecules possessing a C-P bond, such as phosphonates and phosphinates, remain vastly underexplored. Although success stories like fosfomycin have reinvigorated small molecule phosphonate discovery efforts, bioinformatic analyses predict an enormous unexplored biological reservoir of C-P bond-containing molecules, including those attached to complex macromolecules. However, high polarity, a lack of chromophores, and complex macromolecular association impede phosphonate discovery and characterization. Here we detect widespread transcriptional activation of phosphonate biosynthetic machinery across diverse bacterial phyla and describe the use of solid-state nuclear magnetic resonance to detect C-P bonds in whole cells of representative Gram-negative and Gram-positive bacterial species. These results suggest that phosphonate tailoring is more prevalent than previously recognized and set the stage for elucidating the fascinating chemistry and biology of these modifications.

    View details for PubMedID 29068202

  • Whole Ribosome NMR: Dipolar Couplings and Contributions to Whole Cells. The journal of physical chemistry. B Nygaard, R., Romaniuk, J. A., Rice, D. M., Cegelski, L. 2017; 121 (40): 9331-9335


    Solid-state NMR is a powerful tool for quantifying chemical composition and structure in complex assemblies and even whole cells. We employed N{P} REDOR NMR to obtain atomic-level distance propensities in intact 15N-labeled E. coli ribosomes. The experimental REDOR dephasing of shift-resolved lysyl amine nitrogens by phosphorus was comparable to that expected from a calculation of N-P distances involving the lysines included in the crystal structure coordinates. Among the nitrogen contributions to the REDOR spectra, the strongest dephasing emerged from the dipolar couplings to phosphorus involving nitrogen peaks ascribed primarily to rRNA, and the weakest dephasing arose from protein amide nitrogens. This approach is applicable to any macromolecular system and provides quantitative comparisons of distance proximities between shift-resolved nuclei of one type and heteronuclear dephasing spins. Enhanced molecular specificity could be achieved through the use of spectroscopic filters or specific labeling. Furthermore, ribosome 13C and 15N CPMAS spectra were compared with those of whole cells from which the ribosomes were isolated. Whole-cell signatures of ribosomes were identified and should be of value in comparing overall cellular ribosome content in whole-cell samples.

    View details for DOI 10.1021/acs.jpcb.7b06736

    View details for PubMedID 28901760

    View details for PubMedCentralID PMC5897578

  • Disentangling Nanonets: Human a-Defensin 6 Targets Candida albicans Virulence. Biochemistry Cegelski, L. 2017; 56 (8): 1027-1028

    View details for DOI 10.1021/acs.biochem.7b00062

    View details for PubMedID 28198610

  • Mechanochemical unzipping of insulating polyladderene to semiconducting polyacetylene Science Chen, Z., Mercer, J. A., Zhu, X., Romaniuk, J. A., Pfattner, R., Cegelski, L., Martinez, T. J., Burns, N. Z., Xia, Y. 2017; 357 (6350): 475-479


    Biological systems sense and respond to mechanical stimuli in a complex manner. In an effort to develop synthetic materials that transduce mechanical force into multifold changes in their intrinsic properties, we report on a mechanochemically responsive nonconjugated polymer that converts to a conjugated polymer via an extensive rearrangement of the macromolecular structure in response to force. Our design is based on the facile mechanochemical unzipping of polyladderene, a polymer inspired by a lipid natural product structure and prepared via direct metathesis polymerization. The resultant polyacetylene block copolymers exhibit long conjugation length and uniform trans-configuration and self-assemble into semiconducting nanowires. Calculations support a tandem unzipping mechanism of the ladderene units.

    View details for DOI 10.1126/science.aan2797

  • Pseudomonas phage inhibition of Candida albicans. Microbiology (Reading, England) Nazik, H. n., Joubert, L. M., Secor, P. R., Sweere, J. M., Bollyky, P. L., Sass, G. n., Cegelski, L. n., Stevens, D. A. 2017


    Pseudomonas aeruginosa (Pa) and Candida albicans (Ca) are major bacterial and fungal pathogens in immunocompromised hosts, and notably in the airways of cystic fibrosis patients. The bacteriophages of Pa physically alter biofilms, and were recently shown to inhibit the biofilms of Aspergillus fumigatus. To understand the range of this viral-fungal interaction, we studied Pa phages Pf4 and Pf1, and their interactions with Ca biofilm formation and preformed Ca biofilm. Both forms of Ca biofilm development, as well as planktonic Ca growth, were inhibited by either phage. The inhibition of biofilm was reversed by the addition of iron, suggesting that the mechanism of phage action on Ca involves denial of iron. Birefringence studies on added phage showed an ordered structure of binding to Ca. Electron microscopic observations indicated phage aggregation in the biofilm extracellular matrix. Bacteriophage-fungal interactions may be a general feature with several pathogens in the fungal kingdom.

    View details for PubMedID 28982395

  • Visualization of Aspergillus fumigatus biofilms with Scanning Electron Microscopy and Variable Pressure-Scanning Electron Microscopy: A comparison of processing techniques JOURNAL OF MICROBIOLOGICAL METHODS Joubert, L., Ferreira, J. A., Stevens, D. A., Nazik, H., Cegelski, L. 2017; 132: 46-55


    Aspergillus fumigatus biofilms consist of a three-dimensional network of cellular hyphae and extracellular matrix. They are involved in infections of immune-compromised individuals, particularly those with cystic fibrosis. These structures are associated with persistence of infection, resistance to host immunity, and antimicrobial resistance. Thorough understanding of structure and function is imperative in the design of therapeutic drugs. Optimization of processing parameters, including aldehyde fixation, heavy metal contrasting, drying techniques and Ionic Liquid treatment, was undertaken for an ultrastructural approach to understand cellular and extracellular biofilm components. Conventional and Variable Pressure Scanning Electron Microscopy were applied to analyze the structure of biofilms attached to plastic and formed at an air-liquid interface.

    View details for DOI 10.1016/j.mimet.2016.11.002

    View details for Web of Science ID 000393017100009

    View details for PubMedID 27836634

  • Influence of the amyloid dye Congo red on curli, cellulose, and the extracellular matrix in E. coli during growth and matrix purification. Analytical and bioanalytical chemistry Reichhardt, C., McCrate, O. A., Zhou, X., Lee, J., Thongsomboon, W., Cegelski, L. 2016; 408 (27): 7709-7717


    Microbial biofilms are communities of cells characterized by a hallmark extracellular matrix (ECM) that confers functional attributes to the community, including enhanced cohesion, adherence to surfaces, and resistance to external stresses. Understanding the composition and properties of the biofilm ECM is crucial to understanding how it functions and protects cells. New methods to isolate and characterize ECM are emerging for different biofilm systems. Solid-state nuclear magnetic resonance was used to quantitatively track the isolation of the insoluble ECM from the uropathogenic Escherichia coli strain UTI89 and understand the role of Congo red in purification protocols. UTI89 assembles amyloid-integrated biofilms when grown on YESCA nutrient agar. The ECM contains curli amyloid fibers and a modified form of cellulose. Biofilms formed by UTI89 and other E. coli and Salmonella strains are often grown in the presence of Congo red to visually emphasize wrinkled agar morphologies and to score the production of ECM. Congo red is a hallmark amyloid-binding dye and binds to curli, yet also binds to cellulose. We found that growth in Congo red enabled more facile extraction of the ECM from UTI89 biofilms and facilitates isolation of cellulose from the curli mutant, UTI89ΔcsgA. Yet, Congo red has no influence on the isolation of curli from curli-producing cells that do not produce cellulose. Sodium dodecyl sulfate can remove Congo red from curli, but not from cellulose. Thus, Congo red binds strongly to cellulose and possibly weakens cellulose interactions with the cell surface, enabling more complete removal of the ECM. The use of Congo red as an extracellular matrix purification aid may be applied broadly to other organisms that assemble extracellular amyloid or cellulosic materials. Graphical abstract Solid-state NMR was used to quantitatively track the isolation of the insoluble amyloid-associated ECM from uropathogenic E. coli and understand the role of Congo red in purification protocols.

    View details for PubMedID 27580606

  • Fungal biofilm composition and opportunities in drug discovery. Future medicinal chemistry Reichhardt, C., Stevens, D. A., Cegelski, L. 2016; 8 (12): 1455-1468


    Biofilm infections are exceptionally recalcitrant to antimicrobial treatment or clearance by host immune responses. Within biofilms, microbes form adherent multicellular communities that are embedded in an extracellular matrix. Many prescribed antifungal drugs are not effective against biofilm infections owing to several protective factors including poor diffusion of drugs through biofilms as well as specific drug-matrix interactions. Despite the key roles that biofilms play in infections, there is little quantitative information about their composition and structural complexity because of the analytical challenge of studying these dense networks using traditional techniques. Within this review, recent work to elucidate fungal biofilm composition is discussed, with particular attention given to the challenges of annotation and quantification of matrix composition.

    View details for DOI 10.4155/fmc-2016-0049

    View details for PubMedID 27485639

  • Mechanical Behavior of a Bacillus subtilis Pellicle JOURNAL OF PHYSICAL CHEMISTRY B Hollenbeck, E. C., Douarche, C., Allain, J., Roger, P., Regeard, C., Cegelski, L., Fuller, G. G., Raspaud, E. 2016; 120 (26): 6080-6088


    Bacterial biofilms consist of a complex network of biopolymers embedded with microorganisms, and together these components form a physically robust structure that enables bacteria to grow in a protected environment. This structure can help unwanted biofilms persist in situations ranging from chronic infection to the biofouling of industrial equipment, but under certain circumstances it can allow the biofilm to disperse and colonize new niches. Mechanical properties are therefore a key aspect of biofilm life. In light of the recently discovered growth-induced compressive stress present within a biofilm, we studied the mechanical behavior of Bacillus subtilis pellicles, or biofilms at the air-liquid interface, and tracked simultaneously the force response and macroscopic structural changes during elongational deformations. We observed that pellicles behaved viscoelastically in response to small deformations, such that the growth-induced compressive stress was still present, and viscoplastically at large deformations, when the pellicles were under tension. In addition, by using particle imaging velocimetry we found that the pellicle deformations were nonaffine, indicating heterogeneous mechanical properties with the pellicle being more pliable near attachment surfaces. Overall, our results indicate that we must consider not only the viscoelastic but also the viscoplastic and mechanically heterogeneous nature of these structures to understand biofilm dispersal and removal.

    View details for DOI 10.1021/acs.jpcb.6b02074

    View details for Web of Science ID 000379457200033

    View details for PubMedID 27046510

  • Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy. Eukaryotic cell Reichhardt, C., Ferreira, J. A., Joubert, L., Clemons, K. V., Stevens, D. A., Cegelski, L. 2015; 14 (11): 1064-1072


    Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The (13)C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional (15)N and (31)P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions.

    View details for DOI 10.1128/EC.00050-15

    View details for PubMedID 26163318

    View details for PubMedCentralID PMC4621319

  • Frequency-selective REDOR and spin-diffusion relays in uniformly labeled whole cells SOLID STATE NUCLEAR MAGNETIC RESONANCE Rice, D. M., Romaniuk, J. A., Cegelski, L. 2015; 72: 132-139

    View details for DOI 10.1016/j.ssnmr.2015.10.008

    View details for PubMedID 26493462

  • Cell-Based High-Throughput Screening Identifies Rifapentine as an Inhibitor of Amyloid and Biofilm Formation in Escherichia coli. ACS infectious diseases Maher, M. C., Lim, J. Y., Gunawan, C., Cegelski, L. 2015; 1 (10): 460-8


    Escherichia coli assemble functional amyloid fibers termed curli that contribute to bacterial adhesion, biofilm formation, and host pathogenesis. We developed a cell-based high-throughput screen to identify inhibitors of curli-mediated adhesion in the laboratory strain MC4100 and curli-associated biofilm formation in the uropathogenic E. coli clinical isolate UTI89. Inhibitors of biofilm formation can operate through many mechanisms, and such inhibitors could hold therapeutic value in preventing and treating urinary tract infections. The curli-specific screen allows the identification of compounds that inhibit either curli expression, curli biogenesis, or adhesion by normally produced curli. In screening the NIH Clinical Collection of 446 compounds, we identified rifapentine as a potent inhibitor in both of these screens. Rifapentine is an antibiotic used to treat tuberculosis that targets RNA polymerase, but prevents curli-dependent adhesion and biofilm formation in E. coli at concentrations below those that affect viability. Rifapentine inhibits curli production and prevents biofilm formation on plastic, on agar, and at the air-liquid interface by inhibiting curli gene transcription. Comparisons with a cephalosporin antibiotic further revealed that curli production is not affected by standard antibiotic treatment and cell killing pressure. Thus, we reveal a new role independent of killing activity for rifapentine as an inhibitor of curli and curli-mediated biofilm formation.

    View details for DOI 10.1021/acsinfecdis.5b00055

    View details for PubMedID 27623312

  • C-di-GMP Regulates Motile to Sessile Transition by Modulating MshA Pili Biogenesis and Near-Surface Motility Behavior in Vibrio cholerae. PLoS pathogens Jones, C. J., Utada, A., Davis, K. R., Thongsomboon, W., Zamorano Sanchez, D., Banakar, V., Cegelski, L., Wong, G. C., Yildiz, F. H. 2015; 11 (10)

    View details for DOI 10.1371/journal.ppat.1005068

    View details for PubMedID 26505896

  • Cell-Based High-Throughput Screening Identifies Rifapentine as an Inhibitor of Amyloid and Biofilm Formation in Escherichia coli ACS INFECTIOUS DISEASES Maher, M. C., Lim, J. Y., Gunawan, C., Cegelski, L. 2015; 1 (10): 460-468
  • Bottom-up and top-down solid-state NMR approaches for bacterial biofilm matrix composition. Journal of magnetic resonance Cegelski, L. 2015; 253: 91-97


    The genomics and proteomics revolutions have been enormously successful in providing crucial "parts lists" for biological systems. Yet, formidable challenges exist in generating complete descriptions of how the parts function and assemble into macromolecular complexes and whole-cell assemblies. Bacterial biofilms are complex multicellular bacterial communities protected by a slime-like extracellular matrix that confers protection to environmental stress and enhances resistance to antibiotics and host defenses. As a non-crystalline, insoluble, heterogeneous assembly, the biofilm extracellular matrix poses a challenge to compositional analysis by conventional methods. In this perspective, bottom-up and top-down solid-state NMR approaches are described for defining chemical composition in complex macrosystems. The "sum-of-the-parts" bottom-up approach was introduced to examine the amyloid-integrated biofilms formed by Escherichia coli and permitted the first determination of the composition of the intact extracellular matrix from a bacterial biofilm. An alternative top-down approach was developed to define composition in Vibrio cholerae biofilms and relied on an extensive panel of NMR measurements to tease out specific carbon pools from a single sample of the intact extracellular matrix. These two approaches are widely applicable to other heterogeneous assemblies. For bacterial biofilms, quantitative parameters of matrix composition are needed to understand how biofilms are assembled, to improve the development of biofilm inhibitors, and to dissect inhibitor modes of action. Solid-state NMR approaches will also be invaluable in obtaining parameters of matrix architecture.

    View details for DOI 10.1016/j.jmr.2015.01.014

    View details for PubMedID 25797008

    View details for PubMedCentralID PMC4383093

  • Spectral Snapshots of Bacterial Cell-Wall Composition and the Influence of Antibiotics by Whole-Cell NMR BIOPHYSICAL JOURNAL Nygaard, R., Romaniuk, J. A., Rice, D. M., Cegelski, L. 2015; 108 (6): 1380-1389


    Gram-positive bacteria surround themselves with a thick cell wall that is essential to cell survival and is a major target of antibiotics. Quantifying alterations in cell-wall composition are crucial to evaluating drug modes of action, particularly important for human pathogens that are now resistant to multiple antibiotics such as Staphylococcus aureus. Macromolecular and whole-cell NMR spectroscopy allowed us to observe the full panel of carbon and nitrogen pools in S. aureus cell walls and intact whole cells. We discovered that one-dimensional (13)C and (15)N NMR spectra, together with spectroscopic selections based on dipolar couplings as well as two-dimensional spin-diffusion measurements, revealed the dramatic compositional differences between intact cells and cell walls and allowed the identification of cell-wall signatures in whole-cell samples. Furthermore, the whole-cell NMR approach exhibited the sensitivity to detect distinct compositional changes due to treatment with the antibiotics fosfomycin (a cell-wall biosynthesis inhibitor) and chloramphenicol (a protein synthesis inhibitor). Whole cells treated with fosfomycin exhibited decreased peptidoglycan contributions while those treated with chloramphenicol contained a higher percentage of peptidoglycan as cytoplasmic protein content was reduced. Thus, general antibiotic modes of action can be identified by profiling the total carbon pools in intact whole cells.

    View details for DOI 10.1016/j.bpj.2015.01.037

    View details for Web of Science ID 000351774800011

    View details for PubMedID 25809251

    View details for PubMedCentralID PMC4375516

  • Toward a Biorelevant Structure of Protein Kinase C Bound Modulators: Design, Synthesis, and Evaluation of Labeled Bryostatin Analogues for Analysis with Rotational Echo Double Resonance NMR Spectroscopy JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Loy, B. A., Lesser, A. B., Staveness, D., Billingsley, K. L., Cegelski, L., Wender, P. A. 2015; 137 (10): 3678-3685


    Protein kinase C (PKC) modulators are currently of great importance in preclinical and clinical studies directed at cancer, immunotherapy, HIV eradication, and Alzheimer's disease. However, the bound conformation of PKC modulators in a membrane environment is not known. Rotational echo double resonance (REDOR) NMR spectroscopy could uniquely address this challenge. However, REDOR NMR requires strategically labeled, high affinity ligands to determine interlabel distances from which the conformation of the bound ligand in the PKC-ligand complex could be identified. Here we report the first computer-guided design and syntheses of three bryostatin analogues strategically labeled for REDOR NMR analysis. Extensive computer analyses of energetically accessible analogue conformations suggested preferred labeling sites for the identification of the PKC-bound conformers. Significantly, three labeled analogues were synthesized, and, as required for REDOR analysis, all proved highly potent with PKC affinities (∼1 nM) on par with bryostatin. These potent and strategically labeled bryostatin analogues are new structural leads and provide the necessary starting point for projected efforts to determine the PKC-bound conformation of such analogues in a membrane environment, as needed to design new PKC modulators and understand PKC-ligand-membrane structure and dynamics.

    View details for DOI 10.1021/jacs.5600886

    View details for Web of Science ID 000351420800039

    View details for PubMedID 25710634

    View details for PubMedCentralID PMC4405261

  • Characterization of the Vibrio cholerae extracellular matrix: a top-down solid-state NMR approach. Biochimica et biophysica acta Reichhardt, C., Fong, J. C., Yildiz, F., Cegelski, L. 2015; 1848 (1): 378-383


    Bacterial biofilms are communities of bacterial cells surrounded by a self-secreted extracellular matrix. Biofilm formation by Vibrio cholerae, the human pathogen responsible for cholera, contributes to its environmental survival and infectivity. Important genetic and molecular requirements have been identified for V. cholerae biofilm formation, yet a compositional accounting of these parts in the intact biofilm or extracellular matrix has not been described. As insoluble and non-crystalline assemblies, determinations of biofilm composition pose a challenge to conventional biochemical and biophysical analyses. The V. cholerae extracellular matrix composition is particularly complex with several proteins, complex polysaccharides, and other biomolecules having been identified as matrix parts. We developed a new top-down solid-state NMR approach to spectroscopically assign and quantify the carbon pools of the intact V. cholerae extracellular matrix using ¹³C CPMAS and ¹³C{(¹⁵N}, ¹⁵N{³¹P}, and ¹³C{³¹P}REDOR. General sugar, lipid, and amino acid pools were first profiled and then further annotated and quantified as specific carbon types, including carbonyls, amides, glycyl carbons, and anomerics. In addition, ¹⁵N profiling revealed a large amine pool relative to amide contributions, reflecting the prevalence of molecular modifications with free amine groups. Our top-down approach could be implemented immediately to examine the extracellular matrix from mutant strains that might alter polysaccharide production or lipid release beyond the cell surface; or to monitor changes that may accompany environmental variations and stressors such as altered nutrient composition, oxidative stress or antibiotics. More generally, our analysis has demonstrated that solid-state NMR is a valuable tool to characterize complex biofilm systems.

    View details for DOI 10.1016/j.bbamem.2014.05.030

    View details for PubMedID 24911407

  • NMR spectroscopy for atomistic views of biomembranes and cell surfaces. Biochimica et biophysica acta Cegelski, L., Weliky, D. P. 2015; 1848 (1 Pt B): 201-2

    View details for DOI 10.1016/j.bbamem.2014.10.010

    View details for PubMedID 25304061

    View details for PubMedCentralID PMC4267855

  • Characterization of the Vibrio cholerae extracellular matrix: A top-down solid-state NMR approach. Biochimica et biophysica acta Reichhardt, C., Fong, J. C., Yildiz, F., Cegelski, L. 2015; 1848 (1): 378-383

    View details for DOI 10.1016/j.bbamem.2014.05.030

    View details for PubMedID 24911407

  • Congo Red Interactions with Curli-Producing E. coli and Native Curli Amyloid Fibers. PloS one Reichhardt, C., Jacobson, A. N., Maher, M. C., Uang, J., McCrate, O. A., Eckart, M., Cegelski, L. 2015; 10 (10): e0140388


    Microorganisms produce functional amyloids that can be examined and manipulated in vivo and in vitro. Escherichia coli assemble extracellular adhesive amyloid fibers termed curli that mediate adhesion and promote biofilm formation. We have characterized the dye binding properties of the hallmark amyloid dye, Congo red, with curliated E. coli and with isolated curli fibers. Congo red binds to curliated whole cells, does not inhibit growth, and can be used to comparatively quantify whole-cell curliation. Using Surface Plasmon Resonance, we measured the binding and dissociation kinetics of Congo red to curli. Furthermore, we determined that the binding of Congo red to curli is pH-dependent and that histidine residues in the CsgA protein do not influence Congo red binding. Our results on E. coli strain MC4100, the most commonly employed strain for studies of E. coli amyloid biogenesis, provide a starting point from which to compare the influence of Congo red binding in other E. coli strains and amyloid-producing organisms.

    View details for DOI 10.1371/journal.pone.0140388

    View details for PubMedID 26485271

    View details for PubMedCentralID PMC4618944

  • Molecular determinants of mechanical properties of V. cholerae biofilms at the air-liquid interface. Biophysical journal Hollenbeck, E. C., Fong, J. C., Lim, J. Y., Yildiz, F. H., Fuller, G. G., Cegelski, L. 2014; 107 (10): 2245-2252


    Biofilm formation increases both the survival and infectivity of Vibrio cholerae, the causative agent of cholera. V. cholerae is capable of forming biofilms on solid surfaces and at the air-liquid interface, termed pellicles. Known components of the extracellular matrix include the matrix proteins Bap1, RbmA, and RbmC, an exopolysaccharide termed Vibrio polysaccharide, and DNA. In this work, we examined a rugose strain of V. cholerae and its mutants unable to produce matrix proteins by interfacial rheology to compare the evolution of pellicle elasticity in real time to understand the molecular basis of matrix protein contributions to pellicle integrity and elasticity. Together with electron micrographs, visual inspection, and contact angle measurements of the pellicles, we defined distinct contributions of the matrix proteins to pellicle morphology, microscale architecture, and mechanical properties. Furthermore, we discovered that Bap1 is uniquely required for the maintenance of the mechanical strength of the pellicle over time and contributes to the hydrophobicity of the pellicle. Thus, Bap1 presents an important matrix component to target in the prevention and dispersal of V. cholerae biofilms.

    View details for DOI 10.1016/j.bpj.2014.10.015

    View details for PubMedID 25418293

    View details for PubMedCentralID PMC4241461

  • Putative Hydrogen Bond to Tyrosine M208 in Photosynthetic Reaction Centers from Rhodobacter capsulatus Significantly Slows Primary Charge Separation JOURNAL OF PHYSICAL CHEMISTRY B Saggu, M., Carter, B., Zhou, X., Faries, K., Cegelski, L., Holten, D., Boxer, S. G., Kirmaier, C. 2014; 118 (24): 6721-6732


    Slow, ∼50 ps, P* → P(+)HA(-) electron transfer is observed in Rhodobacter capsulatus reaction centers (RCs) bearing the native Tyr residue at M208 and the single amino acid change of isoleucine at M204 to glutamic acid. The P* decay kinetics are unusually homogeneous (single exponential) at room temperature. Comparative solid-state NMR of [4'-(13)C]Tyr labeled wild-type and M204E RCs show that the chemical shift of Tyr M208 is significantly altered in the M204E mutant and in a manner consistent with formation of a hydrogen bond to the Tyr M208 hydroxyl group. Models based on RC crystal structure coordinates indicate that if such a hydrogen bond is formed between the Glu at M204 and the M208 Tyr hydroxyl group, the -OH would be oriented in a fashion expected (based on the calculations by Alden et al., J. Phys. Chem. 1996, 100, 16761-16770) to destabilize P(+)BA(-) in free energy. Alteration of the environment of Tyr M208 and BA by Glu M204 via this putative hydrogen bond has a powerful influence on primary charge separation.

    View details for DOI 10.1021/jp503422c

    View details for Web of Science ID 000337784100040

    View details for PubMedCentralID PMC4064694

  • Solid-state NMR for bacterial biofilms MOLECULAR PHYSICS Reichhardt, C., Cegelski, L. 2014; 112 (7): 887-894
  • Community behavior and amyloid-associated phenotypes among a panel of uropathogenic E. coli. Biochemical and biophysical research communications Lim, J. Y., Pinkner, J. S., Cegelski, L. 2014; 443 (2): 345-350


    Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infection and engage in a coordinated genetic and molecular cascade to colonize the urinary tract. Disrupting the assembly and/or function of virulence factors and bacterial biofilms has emerged as an attractive target for the development of new therapeutic strategies to prevent and treat urinary tract infection, particularly in the era of increasing antibiotic resistance among human pathogens. UPEC vary widely in their genetic and molecular phenotypes and more data are needed to understand the features that distinguish isolates as more or less virulent and as more robust biofilm formers or poor biofilm formers. Curli are extracellular functional amyloid fibers produced by E. coli that contribute to pathogenesis and influence the host response during urinary tract infection (UTI). We have examined the production of curli and curli-associated phenotypes including biofilm formation among a specific panel of human clinical UPEC that has been studied extensively in the mouse model of UTI. Motility, curli production, and curli-associated biofilm formation attached to plastic were the most prevalent behaviors, shared by most clinical isolates. We discuss these results in the context on the previously reported behavior and phenotypes of these isolates in the murine cystitis model in vivo.

    View details for DOI 10.1016/j.bbrc.2013.11.026

    View details for PubMedID 24239885

    View details for PubMedCentralID PMC3932320

  • Sum of the parts: composition and architecture of the bacterial extracellular matrix. Journal of molecular biology McCrate, O. A., Zhou, X., Reichhardt, C., Cegelski, L. 2013; 425 (22): 4286-4294


    Bacterial biofilms are complex multicellular assemblies that exhibit resistance to antibiotics and contribute to the pathogenesis of serious and chronic infectious diseases. New approaches and quantitative data are needed to define the molecular composition of bacterial biofilms. Escherichia coli biofilms are known to contain polysaccharides and functional amyloid fibers termed curli, yet accurate determinations of biofilm composition at the molecular level have been elusive. The ability to define the composition of the extracellular matrix (ECM) is crucial for the elucidation of structure-function relationships that will aid the development of chemical strategies to disrupt biofilms. We have developed an approach that integrates non-perturbative preparation of the ECM with electron microscopy, biochemistry, and solid-state NMR spectroscopy to define the chemical composition of the intact and insoluble ECM of a clinically important pathogenic bacterium-uropathogenic E. coli. Our data permitted a sum-of-all-the-parts analysis. Electron microscopy revealed supramolecular shell-like structures that encapsulated single cells and enmeshed the bacterial community. Biochemical and solid-state NMR measurements of the matrix and constitutive parts established that the matrix is composed of two major components, curli and cellulose, each in a quantifiable amount. This approach to quantifying the matrix composition is widely applicable to other organisms and to examining the influence of biofilm inhibitors. Collectively, our NMR spectra and the electron micrographs of the purified ECM inspire us to consider the biofilm matrix not as an undefined slime, but as an assembly of polymers with a defined composition and architecture.

    View details for DOI 10.1016/j.jmb.2013.06.022

    View details for PubMedID 23827139

  • REDOR NMR for drug discovery BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Cegelski, L. 2013; 23 (21): 5767-5775


    Rotational-echo double-resonance (REDOR) NMR is a powerful and versatile solid-state NMR measurement that has been recruited to elucidate drug modes of action and to drive the design of new therapeutics. REDOR has been implemented to examine composition, structure, and dynamics in diverse macromolecular and whole-cell systems, including taxol-bound microtubules, enzyme-cofactor-inhibitor ternary complexes, and antibiotic-whole-cell complexes. The REDOR approach involves the integrated design of specific isotopic labeling strategies and the selection of appropriate REDOR experiments. By way of example, this digest illustrates the versatility of the REDOR approach, with an emphasis on the practical considerations of experimental design and data interpretation.

    View details for DOI 10.1016/j.bmcl.2013.08.064

    View details for Web of Science ID 000325483000001

    View details for PubMedID 24035486

  • Curcumin as an amyloid-indicator dye in E. coli. Chemical communications McCrate, O. A., Zhou, X., Cegelski, L. 2013; 49 (39): 4193-4195


    We have demonstrated that curcumin is an amyloid-specific dye in E. coli. Curcumin binds to curliated whole cells and to isolated curli amyloid fibers. Similar to Congo red, curcumin exhibits a red-shift in absorbance and a significant increase in fluorescence upon binding to isolated curli.

    View details for DOI 10.1039/c2cc37792f

    View details for PubMedID 23287899

    View details for PubMedCentralID PMC3633639

  • Disruption of Escherichia coli Amyloid-Integrated Biofilm Formation at the Air-Liquid Interface by a Polysorbate Surfactant LANGMUIR Wu, C., Lim, J. Y., Fuller, G. G., Cegelski, L. 2013; 29 (3): 920-926


    Functional amyloid fibers termed curli contribute to bacterial adhesion and biofilm formation in Escherichia coli . We discovered that the nonionic surfactant Tween 20 inhibits biofilm formation by uropathogenic E. coli at the air-liquid interface, referred to as pellicle formation, and at the solid-liquid interface. At Tween 20 concentrations near and above the critical micelle concentration, the interfacial viscoelastic modulus is reduced to zero as cellular aggregates at the air-liquid interface are locally disconnected and eventually eliminated. Tween 20 does not inhibit the production of curli but prevents curli-integrated film formation. Our results support a model in which the hydrophobic curli fibers associated with bacteria near the air-liquid interface require access to the gas phase to formed strong physical entanglements and to form a network that can support shear stress.

    View details for DOI 10.1021/la304710k

    View details for Web of Science ID 000314082500009

    View details for PubMedID 23259693

    View details for PubMedCentralID PMC3557966

  • REDOR NMR for Drug Discovery Bioorganic and Medicinal Chemistry Letters Cegelski, L. 2013
  • Sum of the Parts: Composition and Architecture of the Bacterial Extracellular Matrix Journal of Molecular Biology McCrate, O. A., Zhou, X., Reichhardt, C., Cegelski, L. 2013
  • Nutrient-Dependent Structural Changes in S. aureus Peptidoglycan Revealed by Solid-State NMR Spectroscopy BIOCHEMISTRY Zhou, X., Cegelski, L. 2012; 51 (41): 8143-8153


    The bacterial cell wall is essential to cell survival and is a major target of antibiotics. The main component of the bacterial cell wall is peptidoglycan, a cage-like macromolecule that preserves cellular integrity and maintains cell shape. The insolubility and heterogeneity of peptidoglycan pose a challenge to conventional structural analyses. Here we use solid-state NMR combined with specific isotopic labeling to probe a key structural feature of the Staphylococcus aureus peptidoglycan quantitatively and nondestructively. We observed that both the cell-wall morphology and the peptidoglycan structure are functions of growth stage in S. aureus synthetic medium (SASM). Specifically, S. aureus cells at stationary phase have thicker cell walls with nonuniformly thickened septa compared to cells in exponential phase, and remarkably, 12% (±2%) of the stems in their peptidoglycan do not have pentaglycine bridges attached. Mechanistically, we determined that these observations are triggered by the depletion of glycine in the nutrient medium, which is coincident with the start of the stationary phase, and that the production of the structurally altered peptidoglycan can be prevented by the addition of excess glycine. We also demonstrated that the structural changes primarily arise within newly synthesized peptidoglycan rather than through the modification of previously synthesized peptidoglycan. Collectively, our observations emphasize the plasticity in bacterial cell-wall assembly and the possibility to manipulate peptidoglycan structure with external stimuli.

    View details for DOI 10.1021/bi3012115

    View details for Web of Science ID 000309805100012

    View details for PubMedID 22974326

    View details for PubMedCentralID PMC3554850

  • Quantitative Analysis of Amyloid-Integrated Biofilms Formed by Uropathogenic Escherichia coli at the Air-Liquid Interface BIOPHYSICAL JOURNAL Wu, C., Lim, J. Y., Fuller, G. G., Cegelski, L. 2012; 103 (3): 464-471


    Bacterial biofilms are complex multicellular assemblies, characterized by a heterogeneous extracellular polymeric matrix, that have emerged as hallmarks of persistent infectious diseases. New approaches and quantitative data are needed to elucidate the composition and architecture of biofilms, and such data need to be correlated with mechanical and physicochemical properties that relate to function. We performed a panel of interfacial rheological measurements during biofilm formation at the air-liquid interface by the Escherichia coli strain UTI89, which is noted for its importance in studies of urinary tract infection and for its assembly of functional amyloid fibers termed curli. Brewster-angle microscopy and measurements of the surface elasticity (G(s)') and stress-strain response provided sensitive and quantitative parameters that revealed distinct stages during bacterial colonization, aggregation, and eventual formation of a pellicle at the air-liquid interface. Pellicles that formed under conditions that upregulate curli production exhibited an increase in strength and viscoelastic properties as well as a greater ability to recover from stress-strain perturbation. The results suggest that curli, as hydrophobic extracellular amyloid fibers, enhance the strength, viscoelasticity, and resistance to strain of E. coli biofilms formed at the air-liquid interface.

    View details for DOI 10.1016/j.bpj.2012.06.049

    View details for Web of Science ID 000307427700011

    View details for PubMedID 22947862

    View details for PubMedCentralID PMC3414876

  • Dimethyl Sulfoxide and Ethanol Elicit Increased Amyloid Biogenesis and Amyloid-Integrated Biofilm Formation in Escherichia coli APPLIED AND ENVIRONMENTAL MICROBIOLOGY Lim, J. Y., May, J. M., Cegelski, L. 2012; 78 (9): 3369-3378


    Escherichia coli directs the assembly of functional amyloid fibers termed "curli" that mediate adhesion and biofilm formation. We discovered that E. coli exhibits a tunable and selective increase in curli protein expression and fiber assembly in response to moderate concentrations of dimethyl sulfoxide (DMSO) and ethanol. Furthermore, the molecular alterations resulted in dramatic functional phenotypes associated with community behavior, including (i) cellular agglutination in broth, (ii) altered colony morphology, and (iii) increased biofilm formation. Solid-state nuclear magnetic resonance (NMR) spectra of intact pellicles formed in the presence of [(13)C(2)]DMSO confirmed that DMSO was not being transformed and utilized directly for metabolism. Collectively, the chemically induced phenotypes emphasize the plasticity of E. coli's response to environmental stimuli to enhance amyloid production and amyloid-integrated biofilm formation. The data also support our developing model of the extracellular matrix as an organized assembly of polymeric components, including amyloid fibers, in which composition relates to bacterial physiology and community function.

    View details for DOI 10.1128/AEM.07743-11

    View details for Web of Science ID 000302807500039

    View details for PubMedID 22389366

    View details for PubMedCentralID PMC3346451

  • Plant Cell-Wall Cross-Links by REDOR NMR Spectroscopy JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Cegelski, L., O'Connor, R. D., Stueber, D., Singh, M., Poliks, B., Schaefer, J. 2010; 132 (45): 16052-16057


    We present a new method that integrates selective biosynthetic labeling and solid-state NMR detection to identify in situ important protein cross-links in plant cell walls. We have labeled soybean cells by growth in media containing l-[ring-d(4)]tyrosine and l-[ring-4-(13)C]tyrosine, compared whole-cell and cell-wall (13)C CPMAS spectra, and examined intact cell walls using (13)C{(2)H} rotational echo double-resonance (REDOR) solid-state NMR. The proximity of (13)C and (2)H labels shows that 25% of the tyrosines in soybean cell walls are part of isodityrosine cross-links between protein chains. We also used (15)N{(13)C} REDOR of intact cell walls labeled by l-[ε-(15)N,6-(13)C]lysine and depleted in natural-abundance (15)N to establish that the side chains of lysine are not significantly involved in covalent cross-links to proteins or sugars.

    View details for DOI 10.1021/ja104827k

    View details for Web of Science ID 000284202200047

    View details for PubMedID 20964382

  • REDOR Applications in Biology: An Overview Encyclopedia of NMR Toke, O., Cegelski, L. John Wiley & Sons, Ltd. 2010
  • Small-molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation NATURE CHEMICAL BIOLOGY Cegelski, L., Pinkner, J. S., Hammer, N. D., Cusumano, C. K., Hung, C. S., Chorell, E., Aberg, V., Walker, J. N., Seed, P. C., Almqvist, F., Chapman, M. R., Hultgren, S. J. 2009; 5 (12): 913-919


    Curli are functional extracellular amyloid fibers produced by uropathogenic Escherichia coli (UPEC) and other Enterobacteriaceae. Ring-fused 2-pyridones, such as FN075 and BibC6, inhibited curli biogenesis in UPEC and prevented the in vitro polymerization of the major curli subunit protein CsgA. The curlicides FN075 and BibC6 share a common chemical lineage with other ring-fused 2-pyridones termed pilicides. Pilicides inhibit the assembly of type 1 pili, which are required for pathogenesis during urinary tract infection. Notably, the curlicides retained pilicide activities and inhibited both curli-dependent and type 1-dependent biofilms. Furthermore, pretreatment of UPEC with FN075 significantly attenuated virulence in a mouse model of urinary tract infection. Curli and type 1 pili exhibited exclusive and independent roles in promoting UPEC biofilms, and curli provided a fitness advantage in vivo. Thus, the ability of FN075 to block the biogenesis of both curli and type 1 pili endows unique anti-biofilm and anti-virulence activities on these compounds.

    View details for DOI 10.1038/nchembio.242

    View details for Web of Science ID 000271756900014

    View details for PubMedID 19915538

    View details for PubMedCentralID PMC2838449

  • Microbial Adhesion Encyclopedia of Microbiology Cegelski, L., Smith, C. L., Hultgren, S. J. Academic Press. 2009; 3: 1–10