(4by) Expanding Symbiotic Nitrogen Fixation

My research interests focus on resolving the minimum requirements of symbiotic nitrogen fixation by engineering non-endosymbiotic bacteria to probe the complex mechanisms of this critical biological process. Ammonia-based fertilizers nourish the crops that feed nearly 50% of the world’s current population. While these synthetic fertilizers have largely enabled the Green Revolution, their production and use have introduced significant environmental impacts. Sustainable innovations are required to maintain nutritional security while reducing this damage. Expanding symbiotic nitrogen fixation to deliver the essential macronutrient to non-leguminous crops, such as cereals, could provide an environmentally-balanced solution to this critical challenge. Starting with the addition of essential symbiotic functionalities to nodulation-minus bacteria, my research could help untangle the required mechanisms for this important and perplexing process and move us closer to sustainable agriculture.

Successful Proposals

USDA NIFA Postdoctoral Fellowship; NSF Molecular and Cellular Bioscience; Nebraska Center for Energy Sciences Research; University of Nebraska Office of Research and Economic Development Revision Award; University of Nebraska Core Facility Grant Program for New Users Award; Nebraska Corn Board (two separate grants); NSF Graduate Research Fellowship; People, Prosperity and the Planet Student Design Competition for Sustainability

Postdoctoral Research

My postdoctoral research has centered on developing the synthetic biology tools that will enable the engineering of the plant-growth promoting Rhodopseudomonas palustris CGA009 to: i) utilize its lignin and carbon dioxide valorizing metabolism, ii) query the bacterium’s response to the Nodule-specific Cysteine-Rich (NCR) peptides produced by the legume Aeschynomene evenia, and iii) induce nodule formation in A. evenia. I developed the second and third focus areas independently as early steps towards by long-term research interests, with the second focus area currently funded by a USDA NIFA Postdoctoral Fellowship.

Rajib Saha in the Department of Chemical and Biomolecular Engineering at the University of Nebraska is my advisor for this work.

Graduate Research

My graduate research focused on uncovering the minimum requirements of prokaryotic nitrogen fixation, which is sensitive to oxygen, in an oxygenic cyanobacteria, the predecessors of chloroplasts. What is learned in cyanobacteria could be applied to these oxygen-evolving organelles, which are responsible for photosynthesis in plants. I created genetic circuits that would initiate the production of the nitrogen-fixing enzyme, nitrogenase, in response to environmental conditions important for the enzyme’s function. Three years of this work was funded by an NSF Graduate Research Fellowship.

Tae Seok Moon in the Department of Energy, Environmental, and Chemical Engineering at Washington University in St. Louis was my advisor for this work.

Research Experience

My experience has centered on understanding and utilizing the unique biochemical capabilities of non-model bacteria. As a graduate student, I designed and built regulators that could precisely and predictably control the production of our enzyme of interest in one of the most well-characterized cyanobacterial species. This emphasis has expanded as a postdoctoral researcher to developing tools that explore and harness the biochemical potential of one of the most metabolically versatile, but not well-characterized, bacteria Rhodopseudomonas palustris. Working with this extraordinary bacterium has yielded multiple surprises, such as multiple chromosomes, the importance of a nicking relaxase in plasmid stability, plasmid methylation requirements, and the relationship between energy availability and the range of aromatic compounds catabolized. These discoveries are being characterized and utilized by myself and the graduate and undergraduate students that I mentor. The experience of diagnosing surprising results and converting them into a valuable outcome has been indispensable to my growth as an academic researcher.

Future Direction

My long-term goal is to determine the minimum requirements of nitrogen-fixing symbiosis. My research as a USDA NIFA Postdoctoral Fellow has allowed me to take the first steps towards that goal. As a faculty member, I will continue to use the more basic Nod factor-independent relationship between the legume Aeschynomene evenia and Bradyrhizobium sp. ORS278 as a template to engineer the closely-related, non-endosymbiotic Rhodopseudomonas palustris. The focus of the research is triggering the signal transduction pathway found in vascular plants that is common to both leguminous and arbuscular mycorrhizal symbiosis. My initial projects will include introducing: i) mevalonate production into R. palustris since mevalonate has been shown to activate the common signal transduction pathway and ii) genes R. palustris is missing from two horizontally acquired islands found in Bradyrhizobium sp. ORS278, and four other closely related photosynthetic, symbiotic nitrogen-fixing bacteria, that are associated with lipopolysaccharide synthesis and the plant’s innate immune response.

Selected Publications

DS Long, CM Immethun, L Vallecilla-Yepez, MR Wilkins, R Saha. One step forward, two steps back: transcriptional advancements and fermentation phenomena in Actinobacillus succinogenes 130Z, PLoS ONE, 16:5 (2021).

E Zamani, TJ Johnson, S Chatterjee, C Immethun, A Sarella, R Saha, SK Dishari. Cationic π-Conjugated Polyelectrolyte Shows Antimicrobial Activity by Causing Lipid Loss and Lowering Elastic Modulus of Bacteria, ACS Appl. Mater. Interfaces. 12:44 (2020).

B Brown, C Immethun, M Wilkins, R Saha. Rhodopseudomonas palustris CGA009 polyhydroxybutyrate production from a lignin aromatic and quantification via flow cytometry, Bioresour. Technol. Rep. 11 (2020).

E Zamani, S Chatterjee, T Changa, C Immethun, A Sarella, R Saha, SK Dishari. Mechanistic Understanding of the Interactions of Cationic Conjugated Oligo- and Polyelectrolytes with Wild-type and Ampicillin-resistant Escherichia coli, Sci Rep. 9:1 (2019).

A Alsiyabi, CM Immethun, R Saha. Modeling the Interplay between Photosynthesis, CO₂ Fixation, and the Quinone Pool in a Purple Non-Sulfur Bacterium, Sci Rep. 9:1 (2019).

CM Immethun and TS Moon. in Synthetic Biology of Cyanobacteria Ch. 13. Synthetic Gene Regulation in Cyanobacteria, 317-355, (Springer, 2018).

CM Immethun, DM DeLorenzo, CM Focht, D Gupta, CB Johnson, TS Moon. Physical, Chemical, and Metabolic State Transcriptional Regulators Expand the Synthetic Biology Toolbox for Synechocystis sp. PCC 6803, Biotechnol Bioeng. 114:7 (2017).

N Wan, DM DeLorenzo, L He, L You, CM Immethun, G Wang, EEK Baidoo, W Hollinshead, JD Keasling, TS Moon, YJ Tang. Cyanobacterial Carbon Metabolism: Fluxome Plasticity and Oxygen Dependence, Biotechnol Bioeng. 114:7 (2017).

CM Immethun, KM Ng, DM DeLorenzo, YC Lee, B Waldron-Feinstein, TS Moon. Oxygen-Responsive Genetic Circuits Constructed in Synechocystis sp. PCC 6803, Biotechnol Bioeng. 113:2 (2016).

BM Berla, R Saha, CM Immethun, CD Maranas, TS Moon, HB Pakrasi. Synthetic Biology of Cyanobacteria: Unique Challenges and Opportunities, Front. Microbio. 4:246 (2013).

Teaching Interests

Knowledge acquired in college covers only a small part of what an engineer will need to know throughout their career. It provides the foundation from which we start solving a problem; yet, the broad, complex and dynamic situations engineers face requires the confidence to think creatively and the ability to direct our own learning. Students’ development of a firm knowledge base from which they have the confidence to solve the variety of problems they will face throughout their careers is my first educational objective. My students tackle realistic problems designed to increase their comfort with resolving underdetermined systems, addressing the complex web of needs and constraints, thinking creatively, and communicating effectively. Practicing an organized and dynamically-controlled thought process in new learning situations is the second educational objective for my students. By assessing the task at hand, evaluating the available resources, planning the approach, applying and monitoring the planned strategies, reflecting on the success of the planned strategies, and revising by adjusting the approach, securing additional resources and sometimes even re-assessing the task, students become self-directed learners. I returned to academia propelled by the desire to help students construct the foundation from which they can make important contributions to the challenges we face as a society. My instructional strategies and assessment methods are designed to support that goal and my educational objectives.

Selected Teaching Experience

Teaching and mentoring students has been an important part of my career. I have mentored 18 graduate and undergraduate students in the lab during my academic career, and have co-authored publications with the majority of them. As a graduate student teaching assistant for Chemical Process Dynamics and Control, I developed the material for an optional weekly recitation section. Students that participated in these sessions showed a much higher level of mastery of the material, scoring an average of 20% higher on the associated test. This led to more than 90% of the students participating in the optional sessions by the end of the semester and me receiving the Graduate Student Teaching award, which is based on student evaluations. I have also served as a student advisor and judge for the International Genetically Engineered Machine (iGEM) Competition at both Washington University in St. Louis and the University of Nebraska. In addition to my work with university students, I have lead workshops for middle school and high school teachers about incorporating synthetic biology into their curriculum and developed activities that covered basic scientific concepts for use in their classrooms. Helping students build a foundation for their careers as scientists and engineers has multiplied the number of problems that I can address and expanded the creative solution space, reinforcing the importance of teaching and mentoring students.

Publication

CM Immethun, T Daher, and R Saha. Applying Blended Learning Techniques: Perspectives from Chemical Engineering Computation, Chem Eng Educ. 53:3 (2019).