Chuck Smallwood

Biography

Dr. Chuck Smallwood has broad expertise investigating and engineering cellular biochemical mechanisms in diverse biosystems including bacteria, cyanobacteria, fungi, plants, and microalgae. His NIH funded doctoral research on biochemical mechanisms of ligand-receptor interactions in iron and bacteriotoxin transport lead to fundamental discoveries in cellular membrane active transport and earned him a feature cover on the Journal of General Physiology in 2014. He then joined the University of California, Berkeley as an NIH funded postdoctoral fellow investigating cellular mechanisms of antibiotic resistance and multi-drug transport in bacterial pathogenesis. His research as a Postdoctoral Research Associate and then Staff Scientist at Pacific Northwest National Laboratory centered on integrated Systems Biology approaches of bioimaging, biochemistry, and multi-omics to produce metabolic models and develop genetic toolkits to engineer carbon and nitrogen metabolism to enable increased cellular lipid biosynthesis in green algae for DOE-BER bioenergy applications.

Education

Bachelor’s Degree: B.S. Biochemistry

Doctoral Degree: Ph.D. Biochemistry

Postdoctoral Fellowships:

University of California, Berkeley

Pacific Northwest National Laboratory

Research Interests

The cellular outer membrane serves as the gatekeeper between the intracellular and extracellular space providing a central essential function to sustain life. Primarily, cell membranes allow for the flow of nutrients into the cell to supply the energy and metabolic precursors for biochemical reactions in cellular pathways. Contrary to nutrient uptake, cell membranes also serve as barriers of protection that sense the environment and protect vulnerable intracellular machinery from hostile attack. However, when toxic small molecules obtain entry through cellular membranes the cell has mechanisms for degradation and expulsion such as multi-drug efflux pumps which are embedded in membranes to facilitate capture and expulsion of unwanted toxins for cellular survival. Most cellular membrane transport mechanisms are multicomponent protein assemblies that are complex in their expression, signaling, and energy transduction. In these contexts, our group utilizes various biochemical and genetic techniques to investigate receptor-mediated signaling in cellular systems for improved drug (i.e. antibiotic) discovery, production of biomaterials, and biotechnology development.

Understanding the complexity of intra- and intercellular mechanisms requires intensive and laborious techniques to obtain valuable quantitative data with meaningful biological context. Moreover, the high number of experiments that provides useful biological data limits the scope of even the most basic studies and in turn impedes the downstream progress and positive impacts on the world. Increasingly, more cost-effective and high-throughput technologies are required for impactful biological research. These high-throughput approaches require the integration of automation robotics combined with advanced learning algorithms to conduct repetitive tasks and untangle the complexity inherent in large datasets. Our research technology focus is to develop high-throughput robotic sampling and biomolecular screening technologies and approaches to enable automated time-resolved bioimaging and biochemical detection technologies to study and engineer various biosystems.

Living organisms are increasingly recognized as biological factories for production of high-value chemical compounds for a range of human applications. Although many biosystems can naturally produce compounds of interest, they are often limited by energy conversion efficiency required for economically viable bioproduction. Therefore, to improve efficiency, fundamental understanding of structural responses, metabolic pathways, and signaling factors involved in production of specific metabolites are required to divert energy sources to desired end-products. High-throughput technologies are excellent at producing large datasets of metabolic responses, but these responses are often complex and missing pieces of connected pathways or required signaling factors. Mesoscale modeling of the dynamic structural and biochemical responses in cellular metabolism can provide a blueprint to map and engineer specific metabolic pathways of interest. Thus, a comprehensive systems wide understanding of cellular biochemical pathways in unique organisms can provide detailed insights to enable metabolic biodesign of living organisms. Combining novel intracellular delivery and genetic toolkits our group is developing synthetic biology approaches to model and divert metabolic pathways for directed applications in human health, biodefense, and bioenergy solutions.

Bioenergetics of Bacterial Iron and Toxin Uptake Mechanisms

To combat bacterial pathogenesis in human hosts we sought to understand iron and toxin ligand uptake in bacteria which involves complex cellular bioenergetic mechanisms. Our approaches initially began with genetic engineering of specific amino acid residue substitutions in the cell surface receptor FepA in over 150 different genetic clones. These constructs led to the development of a novel fluorescence spectroscopic assay that allowed us to probe ligand binding and uptake through cell surface receptors. We also developed monoclonal antibodies to specific outer membrane targets of interest. Combining these genetically engineered constructs and monoclonal antibodies, we developed biochemical assays to probe conformation changes of cell surface receptors. From these experiments we discovered that the iron transport receptor FepA exhibited dynamic motion and induced-fit ligand binding that precedes active transport across the cell outer membrane in gram negative Escherichia coli. Furthermore, FepA induced-fit ligand binding was not dependent on the inner membrane TonB-ExbB-ExbD energy-transducing complex.

Relevant Publications

• Lill, Y.; Jordan, L.D.; Smallwood, C.R.; Newton, S.M.; Lill, M.A.; Klebba, P.E.; Ritchie, K. Confined Mobility of TonB and FepA in Escherichia coli Membranes PLoS One. 2016 Dec 9;11(12) DOI: 10.1371/journal.pone.0160862
• Smallwood C. R., Jordan L. D., Trinh V., Schuerch D., Marco A.G., Hanson M., Shipelskiy Y., Majumdar A., Newton S.M., and Klebba P.E. Concerted Loop Motion Triggers Induced Fit of FepA to Ferric Enterobactin. J Gen Physiol. 2014 (Honored by feature on journal cover.) DOI:10.1085/jgp.201311159
• Jordan L. D., Zhou Y., Smallwood C. R., Lill Y., Ritchie K., Yip W. T., Newton S. M., Klebba P. E. Energy- dependent motion of TonB in the Gram-negative bacterial inner membrane. Proc Natl Acad Sci USA 2013 Jul 9;110(28):11553-8. DOI: 10.1073/pnas.1304243110

Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

There are three general and synergistic mechanisms that pathogenic bacteria utilize to prevent the access of antibiotic drugs and chemotherapeutic agents to their targets 1) enzymatic degradation 2) target mutation and 3) membrane efflux from the bacterial cell. Major contributors to microbial drug resistance are multidrug efflux pumps because they not only circumvent intrinsic and acquired multidrug resistance, but they also mitigate stress responses and facilitate pathogenicity. To characterize transient binding events of various drug compounds we designed extensive site-specific mutations to incorporate ligand-reactive unnatural amino acids within the active drug efflux site of the formative multidrug pump AcrB. Our studies verified molecular dynamic simulations highlighting the versatility of the AcrB multi-drug binding pocket and identifying unique chemical interactions that informed new drug designs. In addition, we developed novel biochemical assays using a Clark electrode-based instrument to measure proton flux and oxygen consumption in living cells devoid of the major efflux pump AcrB in order to probe the extent of ancillary efflux pump contributions to drug resistance.

Relevant Publications

• Vargiu AV, Pos KM, Poole K, Nikaido H. Editorial: Bad Bugs in the XXIst Century: Resistance Mediated by Multi-Drug Efflux Pumps in Gram-Negative Bacteria. Front Microbiol. 2016 May 31;7:833. doi: 10.3389/fmicb.2016.00833
• Kinana AD, Vargiu AV, Nikaido H. Some ligands enhance the efflux of other ligands by the Escherichia coli multidrug pump AcrB. Biochemistry. 2013 Nov 19;52(46):8342-51. doi: 10.1021/bi401303v
• Vargiu AV, Nikaido H. Multidrug binding properties of the AcrB efflux pump characterized by molecular dynamics simulations. Proc Natl Acad Sci U S A. 2012 Dec 11;109(50):20637-42. doi: 10.1073/pnas.1218348109

Integrated Systems Biology for Metabolic Biodesign

Cellular systems are small self-contained biological factories capable of producing useful byproducts from basic nutritional inputs. Microalgae in particular have the potential to produce lipids, carbohydrates, and proteins utilizing salt water, light, and minimal micronutrient inputs. However, the diversity and poorly understood metabolisms of photosynthetic organisms limits our ability to engineer specific metabolic pathways. Thus, we sought to gain a systems level understanding of the simplest photosynthetic eukaryotic microalgae Ostreococcus tauri and examined the cellular structural and dynamic metabolic responses to nitrogen and carbon. This approach began with the DOE-BER funded development of a multimodal Stimulated Raman (SRS) and Coherent Anti-Stokes Raman Scattering (CARS) confocal fluorescence microscope that enabled high-content screening and chemical imaging of metabolites of interest. Combining label-based and label-free imaging approaches allowed us to conduct reductionist studies to understand physiological roles of nitrogen and carbon linkages to starch and lipid (i.e. triacylglycerols) biogenesis. We gained additional higher resolution intracellular structural information with cryogenic soft x-ray tomography at Berkeley National Laboratory’s Advanced Light Source revealing unique structural changes occurring during lipid accumulation. LC-MS/MS multi-omic analysis by Environmental Molecular Science Laboratory at Pacific Northwest National Laboratory provided detailed expression profiles of lipids, proteins, and metabolites. Finally, insights into single cell microalgal responses to environmental stimuli were obtained using high-throughput microfluidic devices and automated time-resolved imaging combined with computational analysis of output datasets to screen phenotypic differences between genetically modified organisms. These integrated approaches were applied to existing databases that produced hole-filling metabolic pathway models and identified targets for genetic engineering for bioproduction applications. These integrated approaches can be applied to a number of different biosystems that will facilitate the development of synthetic biology tools to divert metabolic pathways for metabolite bioproduction of high value targets for directed applications in human health, biodefense, and bioenergy solutions.

Relevant Publications

• Geng, T., Smallwood, C.R., Bredeweg, E.L., Plymale, A.E., Baker, S.E., Evans, J.E., and Kelly, R.T. Multimodal microfluidic platform for controlled culture and analysis of unicellular organisms. 2017 Biomicrofluidics 11 (5), 054104 DOI: 10.1063/1.4986533
• Smallwood, C.R., Chen, J., Kyle, J.E., Chrisler, W. B., Hixson, K.K., Nicora, C.D., Moore, R.J., Purvine, S.O. and Evans, J.E., Integrated systems biology and imaging of the smallest free-living eukaryote Ostreococcus tauri. 2018 bioRXiv DOI: https://doi.org/10.1101/293704 (Under Revision)
• Smallwood, C.R., Chrisler, W., Chen J., Patello E., Boudreau R., McDermott G., Le Gros M., Evans J.E., Ostreococcus tauri is a high-lipid content green algae that extrudes clustered lipid droplets. 2018 bioRXiv (Under Revision) DOI: https://doi.org/10.1101/249052
• Smallwood, C.R., Hill, E.A., Chrisler, W. B., Brookreson, J.T., and Evans, J.E., Optimizing bioreactor growth of the smallest eukaryote 2018 bioRXiv DOI: https://doi.org/10.1101/291211 (Under Revision)

Awards, Honors, and Memberships

Memberships

• American Chemical Society
• American Society for Biochemistry and Molecular Biology
• American Society for Microbiology
• American Society for Cell Biology

Awards and Honors

• Belle W. Goodman for Outstanding Scholarship in Research, 2012
• ASBMB Travel Award, ASBMB 2010 Annual Meeting, Anaheim, CA 2010
• National Science Foundation Research Experience for Undergraduates, 2007

Publications

Showing Results. Show More Publications

Selected Publications

• Henske, J.K., Wilken, S.E., Solomon, K.V., Smallwood, C.R., Shutthanandan, V., Evans, J.E., Theodorou, M.K., O’Malley, M.A. Metabolic characterization of anaerobic fungi provides a path forward for bioprocessing of crude lignocellulose 2017 Biotechnology and bioengineering DOI: 10.1002/bit.26515
• Henske, J. K., Gilmore, S. P . , Knop, D . , Cunningham, F .J. , Sexton, J. A. , Smallwood, C. R. , Shutthanandan, V . , Evans, J. E. , Theodorou, M.K., O’Malley, M. Transcriptomic characterization of Caecomyces churrovis: a novel, non-rhizoid-forming lignocellulolytic anaerobic fungus 2017 Biotechnology for biofuels 10 (1), 305 DOI: 10.1186/s13068-017-0997-4
• Novikova I. V.; Smallwood, C.R; Gong, Y.; Hu, D.; Hendricks, L.; Evans, J.E.; Bhattarai, A.; Hess W.P.; El- Khoury P.Z. Multimodal hyperspectral optical microscopy 2017 Chemical Physics 498, 25-32 DOI: https://doi.org/10.1016/j.chemphys.2017.08.011
• Geng, T., Smallwood, C.R., Bredeweg, E.L., Plymale, A.E., Baker, S.E., Evans, J.E., and Kelly, R.T. Multimodal microfluidic platform for controlled culture and analysis of unicellular organisms. 2017 Biomicrofluidics 11 (5), 054104 DOI: 10.1063/1.4986533 Lill, Y.; Jordan, L.D.; Smallwood, C.R.; Newton, S.M.;
• Lill, M.A.; Klebba, P.E.; Ritchie, K. Confined Mobility of TonB and FepA in Escherichia coli Membranes PLoS One. 2016 Dec 9;11(12) DOI: 10.1371/journal.pone.0160862
• Smallwood C. R., Jordan L. D., Trinh V., Schuerch D., Marco A.G., Hanson M., Shipelskiy Y., Majumdar A., Newton S.M., and Klebba P.E. Concerted Loop Motion Triggers Induced Fit of FepA to Ferric Enterobactin. J Gen Physiol. 2014 (Honored by feature on journal cover.) DOI:10.1085/jgp.201311159
• Jordan L. D., Zhou Y., Smallwood C. R., Lill Y., Ritchie K., Yip W. T., Newton S. M., Klebba P. E. Energy- dependent motion of TonB in the Gram-negative bacterial inner membrane. Proc Natl Acad Sci USA 2013 Jul 9;110(28):11553-8. DOI: 10.1073/pnas.1304243110
• Smallwood, C. R., Marco, A. G., Xiao, Q., Trinh, V., Newton, S. M., and Klebba, P. E. Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB, Mol Microbiol 2009, 72, 1171-1180. DOI: 10.1111/j.1365-2958.2009.06715.x
• Smallwood, C.R., Chen, J., Kyle, J.E., Chrisler, W. B., Hixson, K.K., Nicora, C.D., Moore, R.J., Purvine, S.O. and Evans, J.E., Integrated systems biology and imaging of the smallest free-living eukaryote Ostreococcus tauri. 2018 bioRXiv DOI: https://doi.org/10.1101/293704 (Under Revision)
• Smallwood, C.R., Chrisler, W., Chen J., Patello E., Boudreau R., McDermott G., Le Gros M., Evans J.E., Ostreococcus tauri is a high-lipid content green algae that extrudes clustered lipid droplets. 2018 bioRXiv (Under Revision) DOI: https://doi.org/10.1101/249052
• Smallwood, C.R., Hill, E.A., Chrisler, W. B., Brookreson, J.T., and Evans, J.E., Optimizing bioreactor growth of the smallest eukaryote 2018 bioRXiv DOI: https://doi.org/10.1101/291211 (Under Revision)