Single-molecule Studies of Multicomponent Efflux Complexes and Metal-responsive Regulators in Live Bacteria
Genova, Lauren Alexa
This dissertation has two chapters and two appendices: Chapters 1-2 are published research, and Appendices 1-2 are research in progress. Chapter 1 focuses on biomechanical manipulations of the multicomponent efflux complex, CusCBA, a system used by E. coli to resist copper and silver toxicity. Here, I collaborated with colleagues in Cornell’s mechanical engineering department to investigate the role of mechanical forces on the function of protein complexes that span the envelope of Gram-negative cells. We found that mechanical stress promotes disassembly of the CusCBA complex in E. coli and, as a result, renders bacteria more susceptible to metal toxicity. I performed single-molecule imaging experiments and cell-growth assays, analyzed the data, and wrote the paper, which resulted in a co-first authored publication (Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 25462-25467). Chapter 2 focuses on the two-component regulatory system, CusRS. Here, I collaborated with two post-docs in the Chen Lab to elucidate the mechanism by which sensor CusS interacts with response regulator CusR to aid in E. coli’s metal detoxification. From live cell single-molecule imaging experiments, we determined the timepoint in which CusS-CusR interaction affinity switches on (i.e., after CusS senses its metal substrate) and discovered a substrate-sensing−induced mobilization of CusS. I designed and constructed cell strains, performed biochemical experiments, and helped write the manuscript, in which I am a co-first author (Proc. Natl. Acad. Sci. U.S.A. 2020, 117, doi: 10.1073/pnas.1919816117). The introductions of Appendices 1-2 are patterned from the proposals of Prof. Peng Chen; the rest of these chapters detail my molecular biology progress for these projects. Appendix 1 focuses on a novel bottom-up microbiome approach to understand microbial community and cell-cell interactions in bacterial metal homeostasis. We seek to understand how individual E. coli cells communicate with one another in the context of zinc uptake and efflux and how this communication can be regulated in a cell community under changing zinc environments. Appendix 2 focuses on the tripartite efflux complex EmrAB-TolC, which protects E. coli from ionophores and antibiotics. We plan to elucidate the assembly mechanism of EmrAB-TolC, as well as its substrate-responsive element, to determine whether there are parallels between this system and CusCBA, in which we discovered an adaptor-protein−mediated dynamic pump assembly.
Hernandez, Christopher; Lin, Hening
Chemistry and Chemical Biology
Ph. D., Chemistry and Chemical Biology
Doctor of Philosophy
dissertation or thesis